Method of increasing immunological effect

ABSTRACT

A method of increasing immunological effect in a patient by administering an effective amount of a primary cell derived biologic to the patient, inducing immune production, blocking immune destruction, and increasing immunological effect in the patient. Methods of treating an immune target, treating a tumor, immune prophylaxis, and preventing tumor escape.

BACKGROUND OF THE INVENTION

(1) Field of the Invention

The present invention relates to therapy of the immune system. Inparticular, the present invention relates to a primary cell derivedbiologic and methods of using the same to modify potentiation of theimmune system.

(2) Description of Related Art

In a functioning and competent immune system, immature dendritic cellsingest antigens and migrate to the lymph nodes, where they mature. Theresulting mature dendritic cells are then able to activate naïve Tcells, creating antigen-specific cytotoxic T cells that thenproliferate, enter the circulation, and search out and kill theantigenic target. This is generally a powerful, effective, and fastresponse. For example, the immune system is able to clear out aninfluenza infection between 7-12 days.

Antigenic targets can only be eliminated if the immune system iscompetent. Tumors and various other antigenic targets have effectivelyevolved strategies to successfully evade the host immune system, andvarious molecular and cellular mechanisms responsible for tumor evasionhave been identified. Some of these mechanisms target immune antitumoreffector cells. For example, dysfunction and apoptosis of these cells inthe tumor-bearing host creates an immune imbalance that cannot becorrected by immunotherapies aimed only at activation of anti-tumorimmune responses.

Apoptosis, or Type I cell death, is a type of programmed cell death andcan be induced by stress, infection, or DNA damage. Apoptosis is anintegral process during development; however, in certain instances itcan actually do harm. For example, apoptosis of lymphocyte/hematopoieticpopulations, including T cells, can be a serious problem during cancertherapy-related chemotherapy and/or radiation therapy. These cells tendto be sensitive to chemotherapy and radiation therapy.

There are two major mechanisms controlling apoptosis in the cell, thep53 pathway (pro-apoptotic) and the nuclear factor kappa B (NF-κB)pathway (anti-apoptotic). Both pathways are frequently deregulated intumors, as p53 is usually lost, while NF-κB becomes constitutivelyactive.

Tumor-induced apoptosis of lymphocytes is thought to play a significantrole in the immune suppression seen in cancer patients. Apoptosis ofanti-tumor effector cells has been associated with expression of FasL onthe surface of tumor cells. This is based on well-documented evidencethat Fas/FasL interactions play an important role in the down-modulationof immune functions, including triggering of activation-induced celldeath (AICD), to maintain central and peripheral tolerance. Many humantumors express FasL and can eliminate activated Fas+ effectorlymphocytes via the Fas/FasL pathway. FasL expression on tumor cells hasbeen shown to negatively correlate with patient prognosis. In addition,it has been shown that tumors can release membrane-associated FasLthrough secretion of membranous microvesicles (MVs), thereby providingan explanation for spontaneous apoptosis of T lymphocytes observed inthe peripheral circulation of patients with cancer.

Applicants previously showed that MV detected in sera of patients withoral carcinoma induced caspase-3 cleavage, DNA-fragmentation, cytochromec release, loss of mitochondrial membrane potential (MMP) and TCRζ-chain down-regulation in activated T lymphocytes. Furthermore,Applicants demonstrated that these tumor-derived MV are distinguishablefrom immune cell-derived MV by their unique molecular profile andimmune-suppressive properties. Recent data also indicate that MVs arepresent in sera of patients with squamous cell carcinoma of head andneck (H&NSCC) and that these MVs contain biologically active FasL whichmay be involved in mediating lysis of Fas positive T cells in theperipheral circulation. Thus, the activity of tumor-derived MV mightsignificantly contribute to the dysfunction and death of effector Tcells in cancer patients. The loss of these cells could be responsiblefor inadequate anti-tumor function and, by extension, inadequate immuneresponses to cancer vaccines.

It has been convincingly demonstrated that H&NSCC is able to inducefunctional defects and apoptosis in immune effector cells as well asantigen-presenting cells (APCs) by various mechanisms. In previousstudies, Applicants have observed a high level of apoptosis oftumor-infiltrating lymphocytes (TIL) and T lymphocytes in the peripheralcirculation of H&NSCC and melanoma patients. Applicants demonstratedthat CD8+ T cells are more sensitive to apoptosis than CD4+ cells, andthat the effector and tumor-specific subpopulations of CD8+ T cells arepreferentially targeted for apoptosis. Also, individuals with HIVgenerally experience immune suppression caused by dramatic reductions inhelper T cell populations. This reduction is caused by apoptosis of theHIV-infected helper T cells.

The mechanisms responsible for immune cell dysfunction in patients withcancer are numerous and varied. In addition to a wide variety of solubleimmunosuppressive factors such as PGE2, TGF-β, IL-10, and VEGF, andpro-apoptic ligands such as FasL (described above) that are released bytumor cells or other cells in the tumor microenvironment, suppressorcell populations, i.e., regulatory T cells (T regs), have been shown toplay a key role in down-regulation of anti-tumor host immunity.

Collectively, these mechanisms create a poisonous environment, whichexplains the failure of immunotherapy approaches in the past. In orderto have an effective therapeutic outcome, these tumor-induced mechanismsof immune suppression must be directly addressed. With the newfoundknowledge of the multiple causes of immune dysfunction seen in cancerpatients, it is becoming more apparent that multiple active componentsare needed to create an effective cancer immunotherapy. However, therehave been many difficulties in finding an effective immunotherapy andunderstanding its mechanism of action.

Since toxin-induced tumor regressions of human cancer achieved byWilliam Coley early in the 20^(th) century, cancer therapists haveemployed hundreds of different immune therapies with only relativelyrare clinical responses. Because there was little or no insight into thecause of these failures, no consistent mechanism of action emerged. Inorder to establish a clear mechanism of action, a therapy needed to bedevised which could consistently produce a response that could then bedissected.

Head and neck squamous cell cancer (H&NSCC) offers a good model sincemuch is known about the immune defects seen in these patients. Theyinclude, to name a few, (Whiteside, 2001; Hadden, 1995): 1) T lymphocyteanergy and depletion induced by tumor and host-mediated mechanismincluding prostaglandins, T regs, myeloid suppressor cells,antigen-antibody complexes, and cytokines such as IL-10; 2)monocyte/macrophage functional defects with evidence of suppressor andinflammatory changes (Mantovani, 2002); and 3) dendritic cell (DC)defects characterized by sinus histiocytosis (SH) (Dunn, 2005).

Effective therapeutic efforts were needed to reverse these multipledefects. An extensive review of the literature (Hadden, 1995) and aseries of pre-clinical experiments resulted in the primary cell-derivedbiologic (also known as IRX-2) protocol. The IRX-2 protocol, shown inFIG. 1, employs an initial dose of low dose cyclophosphamide (CY) (300mg/m²) by intravenous infusion to reverse suppression by T regs andperhaps other forms of suppressors. The CY is followed by 10-20 dailyinjections of IRX-2 at the base of the skull to feed into the jugularchains of lymph nodes regional to the cancer.

IRX-2 was originally thought to act via increasing T lymphocyte numberand function. Recent evidence indicates that reversal of tumor-inducedapoptosis is also a major mechanism, as disclosed in U.S. ProvisionalPatent Application No. 60/990,759 to Signorelli, et al. Indomethacin(INDO) was administered daily for approximately 21 days to blockprostaglandin production by tumor and monocyte/macrophages, a knowncancer related suppression mechanism. Zinc was also administered asanother aspect of the immunorestorative component of the strategy(Hadden, 1995).

Additionally, at the time the protocol was developed, the critical roleplayed by dendritic cells as presenters of tumor antigen to T cells wasunknown. It was also unknown that sinus histiocytosis (SH) reflected aDC defect, and specifically a tumor induced failure of maturation andantigen presentation. Mechanism of action studies disclosed in U.S. Pat.Nos. 6,977,072 and 7,153,499 to Applicants made it clear that the IRX-2protocol reverses this DC defect and produces changes in regional lymphnodes which reflect a potent immunization (Meneses, 2003). Morespecifically, these patents disclose a method of inducing the productionof naïve T cells and restoring T cell immunity by administration ofIRX-2, which preferably includes the cytokines IL-1β, IL-2, IL-6, IL-8,INF-γ, and TNF-α. This was one of the first showings that adult humanscan generate naïve T cells through molecular therapy. The presence ofnaïve T cells available for antigen presentation was important in therestoration of immunity.

The mechanistic hypothesis that underpins IRX-2 is similar to that of atherapeutic cancer vaccine, although no exogenous antigen is required.When administered into the neck, the agent is thought to act in thecervical lymph node chain directly on DCs to promote their maturationand subsequent ability to present endogenous tumor antigen to naïve Tcells.

Non-clinical data regarding the mechanism of action of IRX-2 has shownthat the agent effectively stimulates and activates humanmonocyte-derived DCs (Egan, 2007). IRX-2 treatment of immature DCsincreased expression of CD83 and CCR7 (markers for maturation and lymphnode migration, respectively), as well as differentiation molecules thatare important for antigen presentation to naïve T cells. Additionally,IRX-2 induces CD40, CD54, and CD86, which are co-stimulatory receptorsthat are critical for activation of naïve T cells. Functional changes inIRX-2-treated DCs included an increase in antigen presentation and Tcell activity. Taken collectively, IRX-2 treatment of immature DC drivesmorphologic, phenotypic, and functional changes that are consistent withthe development of mature and activated DCs that are able to effectivelystimulate naïve T cells.

In contrast to defined antigen-based therapeutic cancer vaccines whereantigen-specific reactivity can be measured, rejection antigens have notbeen discovered in H&NSCC, thus limiting the ability to measureantigen-specific reactivity after IRX-2 therapy.

While IRX-2 was shown to increase T lymphocyte function and generate newimmature T cells, there was no disclosure or suggestion and thus noconclusive demonstration that IRX-2 prevented apoptosis of those T cellsonce generated and it was not known what the function of the T cellswere after presentation of antigen. There were no experimental resultsthat showed that apoptosis of T cells was prevented or would evensuggest the mechanism of action. Proliferation and apoptosis areseparate cellular processes and it would be imprudent to assume that afactor that causes proliferation would necessarily protect fromprogrammed cell death. The exact mechanism by which IRX-2 restores theantitumor response of T cells, and prevents their apoptosis, was neitherexpressly nor inherently disclosed in the prior art. Furthermore, whileIRX-2 was shown to be effective in the mechanisms described above duringcancer treatment, there has been no evidence that IRX-2 provides thesame mechanism of action in other instances of immune suppressionbesides cancer.

Not only have individual cytokines not been able to completely restoreeach part of the immune system through the promotion of DC maturation,the generation of new T cells, and prevention of their apoptosis; butother therapeutics including multiple cytokines have not been able to dothis as well. For example, MULTIKINE® (Cel-Sci) is effective only on thetumor itself, affecting the cell cycle of the tumor cells. PROVENCE®(sipuleucel-T, Dendreon), GVAX® (Cell Genesys), PROMUNE® (ColeyPharmaceutical Group), Dynavax TLR 9 ISS, ONCOPHAGE® (vitespen,Antigenics), CANVAXIN® (CancerVax), and TROVAX® (Oxford BioMedica) havebeen able to show antigen amplification, dendritic cell processing, andsome cellular adjuvancy. TREMELIMUMAB® (Pfizer) and IPILIMUMAB® (Medarexand Bristol-Myers Squibb) only target the T regulatory cell population.

In addition, some therapeutic agents have addressed the issue ofapoptosis of cells. There are several biological agents and smallmolecules that have been developed to prevent cellular and lymphocyteapoptosis. For example, International Patent Application PublicationWO/2006/039545 to Maxim Pharmaceuticals, Inc. discloses theadministration of a PARP-1 inhibitor and additionally an inhibitor ofreactive oxygen metabolite (ROM) production or release to protecttumorcidal lymphocytes, including cytotoxic T lymphocytes and NK cells,from apoptosis. A cytotoxic lymphocyte stimulatory composition includingvarious cytokines can be co-administered. This application reports thatfree radicals produced by tumor adjacent phagocytes cause dysfunctionand apoptosis in tumorcidal or cytotoxic lymphocytes.

International Patent Application Publication WO/2005/056041 to ClevelandClinic Foundation discloses latent TGF-β as a compound that can be usedto protect a patient from treatments that induce apoptosis. The latentTGF-β induces NF-κB activity, thus preventing apoptosis.

International Patent Application Publication WO/2007/060524 to Fundacionde la Comunidad Valenciana discloses various ringed compounds that areinhibitors of Apaf-1 and therefore act as apoptosis inhibitors. Apaf-1is an apoptotic protease-activating factor that makes up part of anapoptosome. Capsase-9 is activated within the apoptosome and initiatesapoptotic signals.

Amifostine (ETHYOL, MedImmune) is another compound that is administeredin order to reduce toxicities resulting from chemotherapy andradiotherapy. More specifically, it is an intravenous organicthiophosphate cytoprotective agent.

There are several disadvantages to these present treatments. Forbiological agents, there is the problem of difficulty in manufacturingand possible difficulty in specifically targeting a given cellpopulation. For small molecules, there may be a problem of toxicity ifused systemically. Further, agents with a single mechanism of actionhave shown a lack of efficacy because multiple activities are needed topromote anti-apoptotic effects in lymphocyte cell populations. Also,none of these treatments directly address the immunosuppressiveenvironment created by the tumor. Thus, effective adjuvants andapproaches to neutralize the tumor-induced suppression are lacking inthe prior art.

In essence, the earlier work of Applicants described the mechanism ofaction of the primary cell derived biologic with respect to DCmaturation and generation of naïve T cells, i.e. several specific levelsof affecting the immune system. Presented herein is evidence of anotherlevel of effect, of the primary cell derived biologic, namely promotionof the survival of lymphocytes. The data herein, taken together withprior disclosures by the Applicants, show that the primary cell derivedbiologic has a corrective and positive effect on the generation andactivation of specific effectors, and their subsequent survival—eachlevel of the immune system, i.e. each arm of the immune system.Compositions of the prior art are directed to only one of these levels.

Therefore, there is a need for a composition that can effectivelyenhance both effector generation and effector survival and target eacharm of the immune system to restore the immune system and provide acomplete mechanism of action against immune suppression.

BRIEF SUMMARY OF THE INVENTION

The present invention provides for a method of increasing immunologicaleffect, including the steps of administering an effective amount of aprimary cell derived biologic to a patient, inducing immune production,blocking immune destruction, and increasing immunological effect in thepatient.

The present invention provides for a method of treating an immune targetin a patient, including the steps of administering an effective amountof the primary cell derived biologic to the patient, inducing immuneproduction, blocking immune destruction, and treating the immune targetin the patient.

The present invention also provides for a method of treating a tumor ina patient, including the steps of administering an effective amount ofthe primary cell derived biologic to the patient, inducing immuneproduction, blocking immune destruction, and treating the tumor in thepatient.

The present invention further provides for a method of immuneprophylaxis, including the steps of administering an effective amount ofa primary cell derived biologic to a patient, inducing immuneproduction, blocking immune destruction, and preventing immunesuppression in the patient.

The present invention also provides for a method of preventing tumorescape in a patient, including the steps of administering an effectiveamount of a primary cell derived biologic to the patient, inducingimmune production, blocking immune destruction, and preventing tumorescape in the patient.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

Other advantages of the present invention will be readily appreciated asthe same becomes better understood by reference to the followingdetailed description when considered in connection with the accompanyingdrawings wherein:

FIG. 1 is a display of the IRX-2 protocol;

FIG. 2 is a representation of the mechanism of action of IRX-2 incombination with a chemical inhibitor and NSAID;

FIG. 3 is a representation of the mechanism of action of IRX-2;

FIG. 4A shows CD8+ Jurkat cells analyzed for Annexin V binding by flowcytometry, FIG. 4B shows Caspase activation detected by FITC-VAD-FMKstaining and flow cytometry; FIG. 4C shows mean percentage+/−standarddeviation (SD) of Annexin V-positive/7-AAD-negative Jurkat cellsfollowing incubation with various apoptosis-inducing agents, and FIG. 4Dshows mean percentage+/−SD of FITC-VAD-FMK+ Jurkat T cells followingincubation with various apoptosis-inducing agents;

FIG. 5A shows a time-course analysis of CD8+ Jurkat cells, and FIG. 5Bshows a concentration-course analysis;

FIG. 6 shows a graph of percentage of FITC-VAD-FMK positive cells forvarious treatments of CD8+ Jurkat cells;

FIG. 7 is a graph of Caspase-activation in Jurkat CD8+ cells aftertreatment with IRX-2 or cytokines and incubation withtumor-microvesicles (MV);

FIG. 8A is a graph of human peripheral blood pre-activated CD4+ cellsand FIG. 8B is a graph of human peripheral blood pre-activated CD8+cells treated with tumor-MV (15 μg) and pre-treated with the indicatedcytokines or IRX-2;

FIG. 9A is a graph of activated human peripheral blood pre-activatedCD4+ cells and FIG. 9B is a graph of human peripheral bloodpre-activated CD8+ cells treated with CH-11 Ab (400 ng/mL) followingpre-treatment with the indicated cytokines or IRX-2;

FIG. 10A shows activation of caspases-3 and 7 in CD8+ Jurkat cellsassessed via flow cytometry for caspase 3/7-FAM binding, and FIG. 10Bshows Western immunoblots showing caspase-3 activation in CD8+ Jurkatcells;

FIG. 11A shows CD8+ Jurkat cells were analyzed by flow cytometry for adecrease in red fluorescence of the cationic dye JC-1, indicating a lossof MMP, and FIG. 11B is a graph of percentage of JC-1 red-negativecells;

FIG. 12A is a fluorescent microscopy of CD8+ Jurkat cells which wereeither untreated (a), incubated for 24 hours with IRX-2 alone (b) or MValone for 24 hours (c) or pre-incubated with IRX-2 for 24 hours andsubsequently treated with MV for 24 hours (d) and then stained by theTUNEL method to reveal DNA strand breaks (red nuclei) indicative ofapoptosis, and FIG. 12B is a graph of percentage of TUNEL-positive CD8+Jurkat cells in the above co-cultures;

FIG. 13 shows Western blots of CD8+ Jurkat cells with varioustreatments;

FIG. 14A is a graph showing that pretreatment of cells with IRX-2reverses the MV-induced changes in the ratios of pro- and anti-apoptoticproteins, and

FIG. 14B is a representative dot plot and corresponding histogramshowing that IRX-2 treatment modulates the expression of pro- andanti-apoptotic proteins;

FIG. 15A is a Western blot of CD8+ Jurkat cells with various treatments,and FIG. 15B is a graph of percentage of FITC-VAD-FMK positive cells;

FIG. 16 is a graph of in vivo dose response for IRX-2;

FIG. 17 is a graph of percentage of survival in four groups of patients;

FIG. 18 is a graph of median percentage of lymphocyte infiltration infour groups of patients;

FIG. 19 is a photograph of H&E staining for lymphocytes;

FIG. 20 is a photograph of H&E staining for lymphocyte infiltration;

FIG. 21A is a graph of lymphoid infiltration density in responders, andFIG. 21B is a graph of lymphoid infiltration density in non-responders;

FIG. 22 is a graph of location of intratumoral/peritumoral lymphocyteinfiltrates;

FIG. 23 is a photograph of IHC staining for CD45RO+ memory T cells;

FIG. 24 is a photograph of fused FDG PET/CT scan images at day 0 and day21;

FIG. 25 is a graph showing Kaplan Meir plots of overall survival;

FIG. 26 is a graph of overall survival for Stage IVa patients;

FIG. 27A is a graph of node size, FIG. 27B is a graph of T cell area,FIG. 27C is a graph of sinus histiocytosis, and FIG. 27D is a graph of Tcell density as compared in controls, H&NSCC controls, and H&NSCCpatients administered IRX-2;

FIG. 28A is a photograph of H&E staining of a typical lymph node in ahead and neck cancer patient with sinus histiocytosis, FIG. 28B is aphotograph of H&E and CD68 staining of a typical lymph node in a headand neck cancer patient with sinus histiocytosis, FIG. 28C is aphotograph of H&E staining of a lymph node with erythrocyte congestionin a head and neck cancer patient with sinus histiocytosis, and FIG. 28Dis graph showing that IRX-2 treatment increases the number of activateddendritic cells in lymph nodes;

FIGS. 29A-C are photographs of H&E staining of tumor samples of patientswith head and neck cancer showing a lack of lymphocyte infiltration;

FIGS. 29D-F are photographs of H&E staining of tumor samples of patientswith head and neck cancer after IRX-2 treatment showing a lack oflymphocyte infiltration;

FIG. 30 is a graph of fibrosis and necrosis in responders andnon-responders;

FIG. 31A is a photograph of tumor fragmentation, FIG. 31B is aphotograph of lymphocyte infiltration, and FIGS. 31C-D are photographsof killer T cells;

FIG. 32 is a display of the mechanism of IRX-2 restoring dendritic cellfunction by up-regulating key activation receptors;

FIG. 33A is a graph of antigen presentation increase (HLA-DR) by IRX-2,and FIG. 33B is a graph of co-stimulation increase (CD86) by IRX-2;

FIG. 34A is a graph of up-regulation of CD40 by IRX-2, and FIG. 34B is agraph of up-regulation of CD54 by IRX-2;

FIG. 35 is a graph of CD83 expression with IRX-2;

FIG. 36 is a graph of dendritic cell-mediatedT cell stimulation afterIRX-2 treatment;

FIG. 37A is a graph of delayed type hypersensitivity and FIG. 37B is agraph of increase in IFN-γ with IRX-2;

FIG. 38 is a graph of delayed type hypersensitivity compared acrosstreatments;

FIG. 39 is a display of evidence of action on immune cells in peripheralcirculation;

FIG. 40 is a graph of T reg counts with IRX-2 treatment; and

FIG. 41A is an image of a tumor pre-treatment with IRX-2, and FIG. 41Bis an image of a tumor post-treatment with IRX-2.

DETAILED DESCRIPTION OF THE INVENTION

In general, the present invention is directed to the application of themechanism of action of IRX-2 both with respect to tumors and the immunesystem in general and provides for a method of treating an immune targetby the administration of a primary cell derived biologic. The primarycell derived biologic produces an immune rejection of the immune target,as further described below by reversing immune incompetence andpotentiating immune response.

More specifically, the primary cell biologic of the present inventionaffects each arm of the immune system (cellular and humoral) and bydoing so the primary cell derived biologic is able to effectivelypotentiate the immune system. The primary cell derived biologic bothinduces immune production and blocks immune destruction, so that thereis a net positive immune effect. In other words, not only are theeffects of immune destruction reversed, but also immune production isincreased notwithstanding that reversal. Thus, the immune system ispotentiated to an even greater degree than if there was increasedhumoral and/or cellular response or only decreased inhibition of immunedestruction. All aspects of the immune system are affected uponadministration of the primary cell derived biologic, as shown in FIG. 3,including dendritic cell maturation, naïve T cell production, effectiveantigen presentation, prevention of apoptosis, and tumor infiltration bylymphocytes. That the primary cell derived biologic has an effect on allaspects of the immune system makes it different and more effective thanprevious therapies, which only address one aspect of the immune system.

The primary cell derived biologic is not a “vaccine” in the classicsense of the word, although it can certainly function in the manner of avaccine. A classic vaccine is used to “turn on” a competent immunesystem and have traditionally been effective only in a prophylacticsetting. The primary cell derived biologic, however, reverses anincompetent immune system, i.e. one in which an immune target or othertherapy (eg radiation, chemotherapy) has rendered incompetent, andreverse this incompetence. In other words, the primary cell derivedbiologic is able to make an immune incompetent patient immune competent.This is a critical step in treating immune targets such as cancer andchronic viral infections.

DEFINITIONS

As used herein, the term “immune target” refers to any antigenic sourceor entity that can be rendered antigenic and afflicts the host patient.Generally, such targets, such as immunogenic pathogens and tumors, showsurface antigen that would otherwise induce an immune response in animmune competent patient. In addition, exogenous antigen can cause anotherwise non-immunogenic immune target to be susceptible to immuneattack in an immune competent patient. With specific regard to thepresent invention, the immune target is immunogenic or potentiallyimmunogenic to which the immune system is nonresponsive due to immuneincompetence from any cause. In the present invention, the immune targetis “targeted” by the immune system made competent by the primary cellderived biologic which reverses the immune suppression and restores theimmune system to function.

The immune incompetence can be caused by genetic defects in thecomponents of the immune system (intrinsic, or primary immunedeficiencies). The immune suppression can also be caused by extrinsicfactors (secondary immune deficiencies). For example, diseases such asAIDS or HIV, irradiation (radiotherapy), chemotherapy, malnutrition,burns, infections, and cancer (tumors) can cause immune suppression.

As used herein, “apoptosis” refers to cell death. As stated above,apoptosis (Type I cell-death) is a type of programmed cell death thatoccurs for various reasons such as stress, infection, or DNA damage.Apoptosis of lymphocytes can be induced by a variety of phenomena, suchas, but not limited to cancer-related therapies (chemotherapy,radiation), and tumors themselves producing apoptosis-inducing factors.

As used herein, “effective amount” refers to an amount of primary cellderived biologic that is needed to achieve the desired result of thepresent invention, namely, protecting lymphocytes and otherhematopoietic components from apoptosis as well as activating the immunesystem to attack an immune target. One skilled in the art can determinethe effective amount of the primary cell derived biologic that should begiven to a particular patient.

As used herein, “increasing immunological effect” refers to the processof changing an incompetent immune system to a competent immune system.The function of a single component of the immune system is reversed fromincompetent to competent, and preferably, the function of multiplecomponents is reversed from incompetent to competent. Therefore, theeffect that the immune system has on an immune target is increased. Acompetent immune system is required to effectively destroy tumors andother immune targets. Not merely turning on but preventing breakdown sothat there is a build up of immunity.

As used herein, “lymphocytes” refers to a white blood cell present inthe immune system and includes large granular lymphocytes (naturalkiller (NK) cells) and small lymphocytes (T cells and B cells).

A “primary cell derived biologic”, as used herein, is a combination ofcytokines, preferably natural and non-recombinant cytokines, alsopreviously known as a natural cytokine mixture (NCM). Preferably, theprimary cell derived biologic is IRX-2 (citoplurikin) as describedbelow, and the two terms can be used interchangeably throughout thisapplication without deviation from the intended meaning.

“IRX-2”, also known as “citoplurikin”, is a leukocyte-derived, naturalprimary cell derived biologic produced by purified human white bloodcells (mononuclear cells) stimulated by phytohemagglutinin (PHA) andciprofloxacin (CIPRO). The major active components are interleukin 1β(IL-1β), interleukin 2 (IL-2), interleukin 6 (IL-6), interleukin 8(IL-8), tumor necrosis factor α (TNF-α), and γ-interferon (IFN-γ).Preferably, the IRX-2 used in the present invention includes these sixcritical cytokines. IRX-2 has also previously been referred to as an“NCM”, a natural cytokine mixture, defined and set forth in U.S. Pat.Nos. 6,977,072 and 7,153,499.

Briefly, IRX-2 is prepared in the continuous presence of a4-aminoquinolone antibiotic and with the continuous or pulsed presenceof a mitogen, which in the preferred embodiment is PHA. Other mitogens,however, can also be used. The IRX-2 produced for administration topatients contains a concentration of IL-1β that ranges from 60-6,000pcg/mL, more preferably, from 150-1,800 pcg/mL; a concentration of IL-2that ranges from 600-60,000 pcg/mL, more preferably, from 3,000-12,000pcg/mL, and concentrations of IFN-γ and TNF-α that range from 200-20,000pcg/mL, more preferably, from 1,000-4,000 pcg/mL.

IRX-2 can also contain a concentration of IL-6 that ranges from 60-6,000pcg/mL, more preferably, from 300-2,000 pcg/mL; a concentration of IL-8that ranges from 6000-600,000 pcg/mL, more preferably from20,000-180,000 pcg/mL; a concentration of TNF-α that ranges from200-20,000 pcg/ml, more preferably, from 1,000-4,000 pcg/mL.Recombinant, natural or pegylated cytokines can be used, or IRX-2 caninclude a mixture of recombinant, natural or pegylated cytokines. TheIRX-2 of the present invention can further include other recombinant,natural or pegylated cytokines such as IL-7, IL-12, IL-15, GM-CSF (at aconcentration that ranges from 100-10,000 pcg/mL, more preferably from500-2,000 pcg/mL), and G-CSF. The method of making IRX-2 is disclosed inthe above cited patents as well as in U.S. Provisional PatentApplication No. 61/044,674.

Other compounds can also be administered along with IRX-2, such aschemical inhibitors, non-steroidal anti-inflammatory drugs (NSAIDS),zinc, and combinations thereof. The chemical inhibitor can be anychemotherapeutic agent (preferably used at low doses) that hasimmunomodulatory effects so as to increase immunity and/or an immuneresponse, e.g., by preferentially inhibiting immune suppression orsuppressor mechanisms in the body. According to a preferred embodiment,the chemical inhibitor is an anti-neoplastic agent, including but notlimited to alkylating agents, antimetabolites and antibiotics. Thechemical inhibitor can also be an immunomodulating agent such asthalidomide. The chemical inhibitor can also be in a salt or othercomplex form. Preferably, the chemical inhibitor is the alkylating agentcyclophosphamide (CY). The NSAID is preferably indomethacin (INDO),which is both a Cox I and Cox II inhibitor. The NSAID can also beibuprofen or Cox II inhibitors such as celecoxib and rofecoxib, orcombinations thereof. The four components used together (i.e. chemicalinhibitor, NSAID, primary cell derived biologic, and zinc) are able toaddress the suppressive environment created by the immune target andrestore the cellular immune response of the patient. More specifically,the chemical inhibitor inhibits T regulatory cells; the NSAID reverseslocal immune suppression by prostaglandins, the primary cell derivedbiologic activates dendritic cells, stimulates T cells, and protects Tcells from apoptosis; and zinc provides key nutrients for T cellfunction as shown in FIG. 2. This combined action encourages immuneresponse to both endogenous and exogenous antigens.

“Tumor escape” as used herein refers to any mechanism by which tumorsescape the host's immune system.

The Overall Mechanism of the Primary Cell Derived Biologic

The present invention is directed to a method of increasingimmunological effect, including the steps of administering an effectiveamount of a primary cell derived biologic, inducing immune production,blocking immune destruction, and increasing immunological effect.

Immune Production

Immune production is induced by maturing immature dendritic cells, theresulting mature dendritic cells activating naïve T cells, modifyingpopulations of B and T cells in blood, activating regional lymph nodes,infiltrating an area adjacent to an immune target with T helper and Bcells, and infiltrating the immune target with T killer cells andmacrophages.

The primary cell derived biologic causes maturation of dendritic cellsand induces the production of naïve T cells, as described in U.S. Pat.Nos. 6,977,072 and 7,153,499. The mature dendritic cells can thenpresent antigen to the naïve and memory T cells and activate them.

The populations of B and T cells can be up-regulated or down-regulated,i.e. modified, due to IRX-2 administration. The populations of B and Tcells in the blood that are modified are more specifically populationsof naïve T cells and early memory T cells. The populations of naïve Tcells that are modified are CD3+ CD45RA+ CCR7+. This is accomplished bydifferentiating naïve T cells into memory and effector T cells, which isa time dependent process. The central memory T cells are also caused toexit the bloodstream and migrate to draining lymph nodes. In otherwords, the modification of levels of naïve T cells is the result of thenaïve T cells differentiating into more advanced forms of T cells thatcan effectively attack the immune target. The populations of B cells inthe blood are also modified because the B cells are recruited into lymphnodes, exposed to antigen, migrate to the immune target, and attack theimmune target. More specifically, the B cells attack the immune targetby producing antibodies and/or supporting antibody-dependent cellularcytotoxicity.

The regional lymph nodes are activated as evidenced by theirenlargement, replenishment of lymphocytes, and reversing sinushistiocytosis. Immunization to antigen to the immune target occurs inthe regional lymph nodes.

Infiltration of the area adjacent to the immune target is mainly byCD45RA+, CD3+, and CD4+ T lymphocytes and CD20+ B lymphocytes. The areaadjacent to the immune target can range from the surface of the immunetarget itself to a distance from the surface. Infiltration of the immunetarget itself, i.e. directly within the immune target, occurs withCD45RO+, CD3+, and CD8+ lymphocytes (i.e. killer T cells) and CD68+macrophages. Each of these infiltration processes contribute toproducing humoral (mediated by antibodies) as well as cellular (mediatedby cells) immunity.

Blocking Immune Destruction

Immune destruction is blocked by protecting the activated T cells fromapoptosis. One of the mechanisms of tumor escape involves targetedelimination of CD8+ effector T cells through apoptosis mediated bytumor-derived microvesicles (MV). Immunosuppressive MV have been foundin neoplastic lesions, sera, ascites and pleural effusions obtained fromcancer patients and have been linked to apoptosis and TCR alterations ineffector T cells in these patients. MV-driven elimination of effector Tcells, which are necessary for anti-tumor host defense, contributes totumor escape and cancer progression. Therefore, protection of anti-tumoreffector cells from functional impairments and death is a majorobjective of immune therapy. Clinical and experimental data show thatcertain cytokines, especially survival cytokines using the commonreceptor γ chain, are able to protect activated T cells fromtumor-induced death and enhance their anti-tumor activity.

More specifically, there are several ways in which IRX-2 protects Tcells from apoptosis. The expression of anti-apoptotic signalingmolecules (i.e. JAK-3 and phosphor-Akt) is up-regulated and theexpression of pro-apoptotic molecules (i.e. SOCS-2) is down-regulated.Activation of caspases in CD8+ and CD4+ T lymphocytes is decreased andcFLIP expression is increased. Inhibition of the PI3K/Akt survivalpathway is counteracted by IRX-2. The T cells are protected from bothextrinsic apoptosis (MV-induced and FasL-induced apoptosis) andintrinsic metabolic (cellular stress or DNA damage related) apoptosis.

The protection from extrinsic MV-induced apoptosis is furtheraccomplished by preventing down-regulation of JAK3, CD3-ζ, and STAT5;inhibiting dephosphorylation of Akt-1/2; and maintaining balanced ratiosof Bax/Bcl-2, Bax-Bcl-xL, and Bim/Mcl-1. The protection from MV-inducedapoptosis is also accomplished by preventing induction of the activityof caspase-3 and caspase-7. More specifically, the induction of theactive cleaved form of caspase-3 is blocked, as is the loss ofmitochondrial membrane potential. Nuclear DNA fragmentation isinhibited. Protection from intrinsic apoptosis by IRX-2 is shown by itsprotection of activated T cells from staurosporine-induced apoptosis.

Importantly, the cytokines of IRX-2 protect the activated T cells fromapoptosis in a synergistic manner. In other words, the combination ofthe cytokines in IRX-2 produces a greater affect than is seen byadministering individual cytokines alone.

The primary cell derived biologic, i.e. IRX-2, administered ispreferably as described above. A chemical inhibitor, low dosecyclophosphamide is preferably administered prior to administering theIRX-2, which reverses suppression by T regs lymphocytes. An NSAID(preferably indomethacin) and zinc can also be administered daily duringthe IRX-2 regimen. Dosing of IRX-2 is further described below.

Inducing immune production and blocking immune destruction isessentially restoring and potentiating the cellular and humoral arms ofa patient's immune system that were previously incompetent. This isaccomplished by restoring naïve T cell populations, activating T and Bcells, promoting infiltration of leukocytes into and adjacent to animmune target, and extending the duration of immune response asdescribed above. These steps of inducing immune production and blockingimmune destruction together produce evidence of increased immunologicaleffect because each arm of the immune system has been changed fromincompetent to competent, and thus immune targets can be effectivelyattacked and destroyed.

Patients who have a suppressed immune system or immune incompetencebenefit from IRX-2 treatment and have their immune system restored tonormal or higher levels of function, i.e. they have a reversal of immuneincompetence and an increased immunological effect. For example, tumorsand other immune targets tend to down-regulate various immune componentsneeded to attack that immune target. Immune targets have defenses whichprevent effective attack by the immune system. Furthermore, thedendritic cells of the immune suppressed patients can induce T and Bcells to become tolerant of the presence of the immune target. Theseimmune targets are susceptible to attack, however, once the immunesystem has been unsuppressed and changed from incompetent to competent.IRX-2 breaks the tolerance by inducing maturation of the dendritic cellsto the immune target, encourages the generation of naïve T cells to beactivated by mature dendritic cells, overcomes suppression, and preventsT cell apoptosis. In this manner, IRX-2 activates each of the arms ofthe immune system as described above in order to overcome all of theprotective effects of the immune target.

Other Embodiments

Various other procedures can be performed in combination with the IRX-2administration in each of the methods of the present invention tofurther enhance therapy such as, but not limited to, surgery,radiotherapy, chemotherapy, or combinations thereof. For example, IRX-2administration before radiotherapy or chemotherapy (cytodestructiveprocesses) improves the results of these processes because IRX-2 acts asa cytoprotectant by protecting T lymphocytes from apoptosis.

The present invention also provides for a method of treating an immunetarget, including the steps of administering an effective amount of aprimary cell derived biologic, inducing immune production and blockingimmune destruction as described above, and treating the immune target.These steps produce evidence of immune rejection of the immune target.In other words, inducing immune protection and blocking immunedestruction is evidence that the immune system has recognized that theimmune target must be destroyed as well as evidence that the immunesystem has been restored to function normally (or at a higher level thanpreviously in a disease or immune suppressed state).

The present invention provides for a method of treating a tumorincluding the steps of administering an effective amount of a primarycell derived biologic, inducing immune production and blocking immunedestruction as described above, and treating the tumor. Morespecifically, the method of treating a tumor is accomplished bymodifying populations of B and T cells in blood, activating regionallymph nodes, peritumorally infiltrating the tumor with T helper and Bcells, intratumorally infiltrating the tumor with killer T cells andmacrophages, and treating the tumor. Killer T cells are produced bymaturing immature dendritic cells, activating naïve T cells, theresulting mature dendritic cells stimulating the naïve T cells, anddifferentiating the naïve T cells into killer T cells. As evidencedherein, the naïve T cells can now differentiate into killer T cells andbecome directed to a tumor so that the tumor can be treated anddestroyed. Each of these steps is as described above. IRX-2 is shownbelow in the Examples to treat tumors in various stages of cancer asevidenced by softening of the tumor, reducing pain caused by the tumor,reducing the size of the tumor, fragmentation of the tumor, necrosis ofthe tumor, and fibrosis of the tumor. In essence, IRX-2 unsuppresses andpotentiates the cellular and humoral arms of immunity so that a tumorcan effectively be treated and cancer eradicated, i.e. completelyeliminated, from a patient.

The present invention provides a method of immune prophylaxis, includingthe steps of administering an effective amount of the primary cellderived biologic, inducing immune production and blocking immunedestruction as described above, and preventing immune suppression.Immune prophylaxis is the prevention of the immune system from beingsuppressed. The primary cell derived biologic actively turns on allparts of the immune system, specifically by maturing immature dendriticcells, activating naïve T cells, the resulting mature dendritic cellsactivating the naïve T cells, protecting the activated naïve T cellsfrom apoptosis (especially when administered before performingchemotherapy or irradiation), differentiating the naïve T cells intomemory and effector T cells, and activating regional lymph nodes so thatthe immune system does not become suppressed. Each of these steps is asdescribed above. If a patient is prone to immune suppression due tobiological factors, this patient can be given IRX-2 preemptively toprevent their immune system from becoming depressed. For example, if apatient has certain genetic factors that predispose them to developingcancer, IRX-2 can be administered so that in the event that an immunetarget such as cancer does become present, the immune system will beready to attack the immune target.

The present invention further provides for a method of preventing tumorescape, including the steps of, administering an effective amount of aprimary cell derived biologic, inducing immune production and blockingimmune destruction as described above, and preventing tumor escape. Morespecifically, tumor escape is prevented by producing an immuneregression of a tumor by modifying populations of B and T cells inblood, activating regional lymph nodes, peritumorally infiltrating thetumor with T helper and B cells, intratumorally infiltrating the tumorwith T killer cells and macrophages. Each of these steps is as describedabove. Many tumors resist immune response by expressingimmunosuppressive signals. Since the immune system is completelyunsuppressed by IRX-2, tumor escape and subsequent metastasis areprevented. Importantly, the patients in the Examples below experienced areduction or delay in recurrence of tumors after IRX-2 treatment,illustrating that IRX-2 can prevent tumor escape.

The present invention provides for a method of protecting activated Tcells from apoptosis, including the steps of administering an effectiveamount of a primary cell derived biologic (IRX-2), and protectingactivated T cells from apoptosis. Essentially, the method of protectingactivated T cells from apoptosis enhances their anti-tumor activity,because the T cells live longer to perform their necessary functions.

The present invention also provides for a method of enhancing theanti-tumor activity of T cells, including the steps of administering aneffective amount of a primary cell derived biologic, stimulating theproduction of naïve T cells, activating the naïve T cells, protectingthe activated T cells from apoptosis, and enhancing the anti-tumoractivity of the T cells. Naïve T cells are produced in response to theadministration of IRX-2, as disclosed in U.S. Pat. Nos. 6,977,072 and7,153,499. These naïve T cells become activated and mature through thepresentation of tumor antigen. According to the present invention, theIRX-2 can now protect these activated T cells from apoptosis. Thisprotection is accomplished as described above.

The present invention is useful in preventing apoptosis in cancerpatients suffering from cancers such as, but not limited to, squamouscell head and neck cancer (H&NSCC), lung, renal-cell, breast, andcolorectal cancers. Furthermore, the present invention can be used toprevent and/or reverse immune suppression in HIV patients by preventingapoptosis of helper T cells.

The present invention also provides for a method of prolonging the lifeof lymphocytes, including the steps of administering an effective amountof a primary cell derived biologic (IRX-2), and prolonging the life oflymphocytes. The lymphocytes that are affected by IRX-2 are preferably Tcells. IRX-2 can further prolong the life of other cells that could beaffected by apoptosis, such as B cells and hematopoietic populations,(dendritic cells, monocytes and myeloid cells). IRX-2 prevents the Tcells from otherwise dying, thus prolonging their lives and allowingthem to acquire and exert the anti-tumor effects for which they areprogrammed, e.g. cytolytic activity or T helper activity. In otherwords, the IRX-2 prevents apoptosis of the T cells, thus prolonging thelives of the T cells.

The present invention further provides for a method of cytoprotectivecancer therapy, including the steps of, administering an effectiveamount of a primary cell derived biologic (IRX-2), producing acytoprotective effect on T cells, and performing a cancer therapy suchas, but not limited to radiation, chemotherapy, and combinationsthereof. Normally, cancer therapies such as radiation and chemotherapysuppress immunity by inducing apoptosis in T cells, i.e. they arecytodestructive. The present invention counteracts the cytodestructiveeffects, preserving immune function and/or accelerating its recoveryafter treatment.

There are several advantages of the present invention with regard toapoptosis. First, the primary cell derived biologic used in the presentinvention is a well-defined biologic that is manufactured in a robustand consistent manner (IRX-2). This is unlike the previously disclosedprior art compounds that are able to protect T cells from apoptosis, butare not manufactured robustly. Further, the cytokines in the primarycell derived biologic used in the present invention act synergisticallyon multiple cell types of the immune system resulting in a coordinatedimmune response at doses far lower than are needed to achieve similarresults using single, recombinant cytokines as monotherapies, asevidenced in the examples below.

As shown in the following examples, it was determined that IRX-2 is ableto protect T cells from apoptosis mediated by tumor-derived MV. Some ofthe cytokines present in the IRX-2 such as IL-2 are known to haveanti-apoptotic effects; thus, it was reasonable to evaluate whether theIRX-2 had protective as well as stimulatory effects on T cells. Thecombined effects of T cell survival enhancement and functionalstimulation underlie IRX-2's apparent synergistic effects in vivo. Usinga previously established in vitro model of tumor-induced apoptosis, itis demonstrated herein that IRX-2 provides strong protection to T cellsfrom apoptosis mediated by tumor-derived MV through activation ofsurvival pathways, thereby effectively counteracting cancer-relatedimmunosuppression. The results of these experiments are presented in theexamples below. Thus, it is shown herein that IRX-2 is effective againsta new arm of the immune system and is able to restore that arm of theimmune system, i.e. preventing apoptosis of lymphocytes.

Tumor-derived microvesicles (MV) expressing a membrane form of FasL werepurified from supernatants of the PCI-13 tumor cell line andco-incubated with CD8+ Jurkat cells or activated peripheral blood (PB) Tcells. FasL, the Fas ligand, is a type II transmembrane proteinbelonging to the tumor necrosis factor (TNF) family. FasL-receptorinteractions play an important role in the regulation of the immunesystem and the progression of cancer. Apoptosis is induced upon bindingand trimerization of FasL with its receptor (FasR), which spans themembrane of a cell targeted for death.

Incubation of CD8+ Jurkat T cells and activated PB T cells withtumor-derived MV induced significant apoptosis, as evidenced byincreased annexin binding (64.4%±6.4), caspase activation (58.1%±7.6), aloss of mitochondrial membrane potential (MMP) (82.9%±3.9) andDNA-fragmentation.

Pre-incubation of T cells with IRX-2 suppressed apoptosis in a dose- andtime-dependent manner (p <0.001 to p <0.005). The observed protectiveeffects on CD8⁺ T cells of IRX-2 were comparable to the cytoprotectiveeffects of recombinant IL-2 or IL-15 alone but was superior to IL-7;however, IRX-2 had greater effects on protecting CD4⁺ T cells fromapoptosis IRX-2 does not contain IL-7 or IL-15 and the fact that it isprotecting CD4⁺ T cells from apoptosis more effectively than equivalentamounts of recombinant IL-2 means that the unique components of IRX-2act synergistically to protect T cells from apoptosis.

IRX-2 suppressed MV-induced down-regulation of JAK3 and theTCR-associated ζ-chain and induced strong Stat5 activation in T cells.Flow cytometry analysis showed that IRX-2 reversed the MV-inducedimbalance of pro- and anti-apoptotic proteins in T cells by suppressingthe MV-mediated up-regulation of pro-apoptotic proteins Bax and Bim (p<0.005 to p <0.05), and concurrently restoring the expression of theanti-apoptotic proteins Bcl-2, Bcl-xL, FLIP and Mcl-1 (p <0.005 to p<0.01). In addition, IRX-2 treatment counteracted the MV-inducedinhibition of the PI3K/Akt survival pathway. A specific Akt-inhibitor(Akti-1/2) abrogated the protective effect of IRX-2, demonstrating thatthe PI3K/Akt pathway plays a key role in IRX-2-mediated survivalsignaling. The PI3K/Akt pathway is a key component in preventingapoptosis and activation of this pathway would prevent against manydifferent inducers of apoptosis. These studies show that a short ex vivopre-treatment with IRX-2 provides potent protection of T cells fromtumor-induced apoptosis. As effector T cells resistant toimmunosuppressive influences of the tumor microenvironment are essentialfor anti-tumor host defense, utilization of IRX-2 significantly improvesthe effectiveness of cancer biotherapies.

The present invention also provides for a method of inducingimmunization in a patient, including the steps of administering aneffective amount of the primary cell derived biologic, detecting achange in T and B cells, and inducing immunization in a patient. Thechanges in circulating T and B cell subset compositions, describedabove, reflect alterations in lymphocyte generation, differentiation,and traffic. i.e. a modification in levels of T cells and B cells inblood occurs because they are differentiating or moving to other areas.These changes in subset composition are evidence that immunization hasbeen induced in a patient.

The present invention also provides a method of predicting a favorabletreatment outcome to cancer treatment, including the steps ofadministering an effective amount of the primary cell derived biologic,detecting an increase peritumorally of T helper and B cells andintratumorally of T killer cells and macrophages, and predicting afavorable treatment outcome to cancer treatment. More specifically, anincrease is detected peritumorally of CD45RA+ CD3+ CD4+ T lymphocytesand CD20+ B lymphocytes and intratumorally of CD45RO+ CD3+ CD8+lymphocytes and CD68+ macrophages as described above. In other words,this characteristic change in leukocyte infiltration is a biomarker thatindicates that treatment with the primary cell derived biologic will beeffective. This biomarker can be used to screen out patients for whomcontinued treatment with the primary cell derived biologic will not besuccessful so that these patients can seek other alternatives. Thismethod can use automated means for predicting the treatment outcome,such as, but not limited to, various assays or immunoassays (ELISA,radioimmunoassays) and high-throughput methods such as flow ormicroscopic cytometry.

Advantages of the Primary Cell Derived Biologic

Overall, IRX-2 unsuppresses and potentiates all aspects of the cellularand humoral arms of the immune system to attack various immune targets.Any immune incompetent disease state (cancer, AIDS, and others aspreviously described above) can now be reversed by unsuppressing andpotentiating the immune system through IRX-2. IRX-2 functions as a“symphony” rather than just a single “instrument” in that the specificcombination of cytokines of IRX-2 effect multiple parts of the immunesystem, as opposed to prior art therapeutics which, while beingcombinations of components, either augment generation of effectors orprevent their apoptosis, i.e. only work on a single part of the immunesystem. Each part of the immune system is a gatekeeper of one effectexperienced by IRX-2 administration. Each of these parts of the immunesystem is required in order to attack an immune target. FIGS. 2 and 3depict the processes potentiated by IRX-2 therapy. Immature dendriticcells must become mature in order to activate naïve T cells. Productionof naïve T cells also must be induced so that they can be presented withantigen by the mature dendritic cells. Both the naïve T cells and thedendritic cells must migrate to the regional lymph node in order forantigen to be presented to the naïve T cells by the dendritic cells.Once activated, the T cells must be protected from apoptosis so thatthey can differentiate into killer T cells and attack the immune target.B cells also mature into plasma cells to aid in attacking the immunetarget. Administration of IRX-2 augments all of these processes,providing a competent immune system that is ready to attack any immunetarget.

Dosing and Administration

Administration of the primary cell derived biologic in vivo is the sameas the vaccine +IRX-2 or IRX-2 alone immunotherapy disclosed in thepreviously mentioned patents related to IRX-2. IRX-2 is preferablyinjected perilymphatically over a 10 day regimen at 115 Units perinjection, but can also be injected with other methods further describedbelow. Alternatively, other regimes can be used wherein the IRX-2 isadministered intermittently. For example, it can be administered threedays a week or five out of seven days a week. As shown below, IRX-2inhibits apoptosis over a range of concentrations: from 1:1 to 1:10dilution of the IRX-2 liquid (i.e. dilution of the IRX-2 in the media inwhich it was produced).

Preferably, the IRX-2 is injected around lymphatics that drain intolymph nodes regional to a lesion, such as a tumor or other persistentlesion. Perilymphatic administration at draining nodes is critical.Peritumoral injection has been associated with little response, increasein the mitotic index of the tumor, even progression and is thuscontraindicated. A ten (10) day injection scheme is optimal and a twenty(20) day injection protocol, while effective clinically, tends to reduceTH1 response and likely shifts towards a less desirable TH2 response asmeasured by lymphoid infiltration into the cancer. Bilateral injectionsare effective. Where radical neck dissection has occurred,contralaterial injection is effective.

The compounds of the present invention (including IRX-2) areadministered and dosed to promote protection from apoptosis as well asoptimal immunization either to exogenous or endogenous antigen, takinginto account the clinical condition of the individual patient, the siteand method of administration, scheduling of administration, patient age,sex, and body weight. The pharmaceutically “effective amount” forpurposes herein is thus determined by such considerations as are knownin the art. The amount is preferably effective to induce immuneproduction and block immune destruction. The amount is also preferablyeffective to promote immunization, leading to, e.g., tumor reduction,tumor fragmentation and leukocyte infiltration, delayed recurrence orimproved survival rate, or improvement or elimination of symptoms.

In the methods of the present invention, the compounds of the presentinvention can be administered in various ways, although the preferredmethod is by perilymphatic injection. It should be noted that thecompounds can be administered as the compounds themselves or as apharmaceutically acceptable derivative and can be administered alone oras an active ingredient in combination with pharmaceutically acceptablecarriers, diluents, adjuvants and vehicles. The compounds can also beadministered intra- or subcutaneously, or peri- or intralymphatically,intranodally or intrasplenically or intramuscularly, intraperitoneally,and intrathoracically. Implants of the compounds can also be useful. Thepatient being treated is a warm-blooded animal and, in particular,mammals including man. The data presented shows activity of the IRX-2 onhumans or cells derived from humans, and therefore the data herein isall directly relevant and applicable to humans. The pharmaceuticallyacceptable carriers, diluents, adjuvants and vehicles as well as implantcarriers generally refer to inert, non-toxic solid or liquid fillers,diluents or encapsulating material not reacting with the activeingredients of the invention.

The doses can be single doses or multiple doses over a period of severaldays, although preferably a 10 day injection scheme is used. Whenadministering the compound of the present invention, it is generallyformulated in a unit dosage injectable form (e.g., solution, suspension,or emulsion). The pharmaceutical formulations suitable for injectioninclude sterile aqueous solutions or dispersions and sterile powders forreconstitution into sterile injectable solutions or dispersions. Thecarrier can be a solvent or dispersing medium containing, for example,water, ethanol, polyol (for example, glycerol, propylene glycol, liquidpolyethylene glycol, and the like), suitable mixtures thereof, andvegetable oils.

Proper fluidity can be maintained, for example, by the use of a coatingsuch as lecithin, by the maintenance of the required particle size inthe case of dispersion and by the use of surfactants. Nonaqueousvehicles such a cottonseed oil, sesame oil, olive oil, soybean oil, cornoil, sunflower oil, or peanut oil and esters, such as isopropylmyristate, can also be used as solvent systems for compoundcompositions. Additionally, various additives which enhance thestability, sterility, and isotonicity of the compositions, includingantimicrobial preservatives, antioxidants, chelating agents, andbuffers, can be added. Prevention of the action of microorganisms can beensured by various antibacterial and antifungal agents, for example,parabens, chlorobutanol, phenol, sorbic acid, and the like. In manycases, it is desirable to include isotonic agents, for example, sugars,sodium chloride, and the like. Prolonged absorption of the injectablepharmaceutical form can be brought about by the use of agents delayingabsorption, for example, aluminum monostearate and gelatin. According tothe present invention, however, any vehicle, diluent, or additive usedwould have to be compatible with the compounds.

Sterile injectable solutions can be prepared by incorporating thecompounds utilized in practicing the present invention in the requiredamount of the appropriate solvent with several of the other ingredients,as desired.

A pharmacological formulation of the present invention can beadministered to the patient in an injectable formulation containing anycompatible carrier, such as various vehicles, additives, and diluents;or the compounds utilized in the present invention can be administeredparenterally to the patient in the form of slow-release subcutaneousimplants or targeted delivery systems such as monoclonal antibodies,vectored delivery, iontophoretic, polymer matrices, liposomes, andmicrospheres. Examples of delivery systems useful in the presentinvention include those disclosed in U.S. Pat. Nos. 5,225,182;5,169,383; 5,167,616; 4,959,217; 4,925,678; 4,487,603; 4,486,194;4,447,233; 4,447,224; 4,439,196; and 4,475,196. Many other suchimplants, delivery systems, and modules are well known to those skilledin the art.

The invention is further described in detail by reference to thefollowing experimental examples. These examples are provided for thepurpose of illustration only, and are not intended to be limiting unlessotherwise specified. Thus, the present invention should in no way beconstrued as being limited to the following examples, but rather, beconstrued to encompass any and all variations which become evident as aresult of the teaching provided herein.

EXAMPLES Materials and Methods

All steps relating to cell culture are performed under sterileconditions. General methods of cellular immunology not described hereinare performed as described in general references for cellular immunologytechniques such as Mishell and Shiigi (Selected Methods in CellularImmunology, 1981) and are well known to those of skill in the art.

Antibodies and Reagents:

The following monoclonal antibodies were used for flow cytometryanalysis: anti-CD3-ECD, -CD8-PCS, -CD4-PE (Beckman Coulter, Miami,Fla.); anti-Bcl-2-FITC, -Bcl-2-PE, -Fas-FITC, -FasL-PE (BD Biosciences,San Jose, Calif.); anti-Bax-FITC, -Bcl-xL-FITC (Santa CruzBiotechnology, Santa Cruz, Calif.) and anti-Bid-antibody (Abcam Inc.,Cambridge, Mass.). Polyclonal antibodies were: anti-Bim (Cell Signaling,Danvers, Mass.), anti-FLIP (GenWay Biotech, San Diego, Calif.), andanti-Mcl-1 (Santa Cruz Biotechnology). FITC-conjugated Annexin V waspurchased from Beckman Coulter. FITC-conjugated anti-rabbit IgG waspurchased from Jackson ImmunoResearch Laboratories (West Grove, Pa.) andthe isotype controls (IgG₁-FITC, IgG_(2a)-FITC and IgG_(2b)-FITC andIgG2-PE) were purchased from BD Biosciences. Antibodies purchased forWestern Blot analysis included: polyclonal phospho-Akt (Ser473),polyclonal total-Akt, monoclonal phospho-STAT5 (Tyr694) and monoclonaltotal-STAT5 (Cell Signaling), monoclonal Bcl-2, monoclonal CD3-ζ,monoclonal JAK3, polyclonal SOCS-2 and polyclonal Mcl-1 (Santa CruzBiotechnology), polyclonal caspase-3, polyclonal FasL antibody-3 (BDBiosciences) and monoclonal β-actin (Sigma Aldrich, St. Louis, Mo.).Anti-Fas (CH-11) agonistic monoclonal antibody, IgM isotype control forCH-11, anti-Fas blocking monoclonal antibody, clone ZB4, and isotypeIgG1 control for ZB4 were all purchased from Upstate Biotechnology (LakePlacid, N.Y.). All cell culture reagents including AIM V medium, RPMI1640 medium, phosphate-buffered saline (PBS), heat-inactivated fetalcalf serum (ΔFCS), streptomycin, penicillin, I-glutamine, recombinanttrypsin-like enzyme (TrypLE) and trypan blue dye were purchased fromGibco/Invitrogen (Grand Island, N.Y.). The human recombinant cytokines,rhIL-2, rhIL-7 and rhIL-15, were purchased from Peprotech Inc. (RockyHill, N.J.). Bovine serum albumin (BSA), saponin, etoposide andstaurosporine were from Sigma Aldrich. 7-amino-actinomycin D (7AAD) andthe pan caspase inhibitor, z-VAD-FMK, were obtained from BD Biosciences.The selective inhibitor of Akt1/Akt2 was purchased from Calbiochem (SanDiego, Calif.) and the selective inhibitors for caspase-3, caspase-8 andcaspase-9 from R&D Systems (Minneapolis, Minn.).

Preparation of Primary Cell Derived Biologic (IRX-2):

The method of making the primary cell derived biologic is generallydescribed in U.S. Provisional Patent Application No. 61/044,674.Mononuclear cells (MNCs) are purified to remove contaminating cells byloading leukocytes onto lymphocyte separation medium (LSM) andcentrifuging the medium to obtain purified MNCs with an automated cellprocessing and washing system. The MNCs are then stored overnight in aFEP lymphocyte storage bag. An induction mixture of the MNCs isstimulated with a mitogen, preferably phytohemagglutinin (PHA), andciprofloxacin in a disposable cell culture device and a primary cellderived biologic is produced from the MNCs. The mitogen is removed fromthe induction mixture by filtering and tangential flow filtration mode,and then the induction mixture is incubated. The induction mixture isclarified by filtering to obtain a primary cell derived biologicsupernatant. Finally, the primary cell derived biologic supernatant iscleared from DNA and adventitious agents by applying anion exchangechromatography and 15 nanometer filtration and optionally furtherinactivation by ultraviolet-C (UVC). The final product can then bevialed and stored for future administration to a patient.

Cells and Cell Lines:

The head and neck squamous cell carcinoma (H&NSCC) cell line PC-13 wasestablished in Applicants' laboratory and maintained as previouslydescribed. It was retrovirally transfected with the human FasL geneobtained from Dr. S. Nagata (Osaka Biosciences Institute, Osaka, Japan)as previously reported. Supernatants of transfected PCI-13-cells(PCI-13-FasL), which contained both sFasL and the 42 kDa membranous formof FasL, were used as a source of tumor-derived microvesicles (MV).Jurkat cells were obtained from American Tissue Culture Collection(ATCC, Manassas, Va.) and were transfected with CD8. The CD8+ Jurkatcells were cultured in RPMI 1640 medium supplemented with 10% (v/v)fetal bovine serum (FBS), L-glutamine and antibiotics. Human Tlymphocytes were isolated from peripheral blood mononuclear cells (PBMC)obtained from consented normal donors. PBMC were isolated byFicoll-Hypaque density gradient centrifugation (GE HealthcareBio-Sciences Corp., Piscataway, N.J.), washed and plated for 1 hour at37° C. in culture flasks (T162) in an atmosphere of 5% CO₂ to removeCD14+ monocytes. The non-adherent T-lymphocyte fraction was collectedand immediately used for experiments or cryopreserved. CD8+ T cells orCD4+ T cells were purified by positive selection using CD8 MicroBeads orCD4 MicroBeads, respectively (Miltenyi Biotec, Auburn, Calif.) using theAutoMACS system according to the manufacturer's instructions. PurifiedCD8+ or CD4+ T cells were then cultured for 2-3 days in AIM V mediumsupplemented with 10% FBS in the presence of beads coated with anti-CD3and anti-CD28 antibodies (T Cell Activation/Expansion Kit, MiltenyiBiotec). All cells used for the above described experiments were in thelog phase of growth.

Isolation of Microvesicles:

Microvesicles (MV) were isolated from culture supernatants of theFasL-transfected PCI-13 cell line as previously described. Briefly, theconcentrated cell culture supernatants were fractioned by a two-stepprocedure, including size exclusion chromatography andultracentrifugation. PCI-13-FasL supernatants were concentrated at least10 times using Centriprep Filters (Fisher Scientific, Pittsburgh, Pa.).Next, 10 mL aliquots of concentrated supernatants were applied to aSepharose 2B (Amersham Biosciences, Piscataway, N.J.) column (1.5×35 cm)equilibrated with PBS. One milliliter fractions were collected and theprotein content was monitored by measuring absorbance at 280 nm. Theexclusion peak material, containing proteins of >50 million kDA, wasthen centrifuged at 105,000×g for 2 hours at 4° C. The pellet wasresuspended in 300-500 μl of sterile PBS. Protein concentration in eachMV preparation was estimated by a Lowry's protein assay (Bio-RadLaboratories, Hercules, Calif.) with bovine serum albumin (BSA) used asa standard.

Western Blot Assays:

To determine total or phosphorylated forms of Akt, Bcl-2, CD3ζ,caspase-3, JAK3, STAT5, FLIP, and Mcl-1, Jurkat CD8+ cells or purifiedactivated CD8+ or CD4+ T-cells were co-incubated with MV at theindicated concentration and/or with IRX-2 (1:3 final dilution) for theindicated period of time at 37° C. The cells were then washed,centrifuged at 4° C. and lysed in equal volumes of ice-cold lysis-buffer(50 mM Tris-HCL pH 7.5, 150 mM NaCl, 0.5% Nonidet P-40) and proteaseinhibitor cocktail (Pierce Chemical Co., Rockford, Ill.). After lysis,the homogenates were clarified by centrifugation. The supernatants wereisolated, and boiled for 5 minutes in 5× Laemmli sample buffer. Proteinswere separated by sodium dodecyl sulfate-polyacrylamide gelelectrophoresis (SDS-PAGE) and electrotransfered to polyvinylidenedifluoride (PVDF) membranes. The membranes were blocked in 5% fat-freemilk or 5% BSA in TTBS (0.05% Tween 20 in Tris-buffered saline) for 1hour at room temperature (RT) and then incubated overnight at 4° C. withthe appropriate antibodies. After washing (3×15 minutes) with TTBS atRT, membranes were incubated with horseradish peroxidase-conjugatedsecondary antibody at 1:150,000 dilution (Pierce Chemical Co) for 1 hourat RT. After washes, membranes were developed with a SuperSignalchemoluminescent detection system (Pierce Chemical Co). To reprobe withanother primary antibody, membranes were incubated in stripping buffer(0.5 M NaCl, 3% (v/v) glacial acetic acid), washed and then used forfurther study.

Co-Incubation of CD8+ Jurkat Cells or Activated Normal T-Lymphocyteswith MV and IRX-2:

CD8+ Jurkat cells or activated normal T lymphocytes were plated at0.3×10⁶ cells per well in a 96-well plate and pre-treated or not withIRX-2 or with recombinant human cytokines at a final concentration of 10ng/mL or 100 IU/mL for 24 hours (unless otherwise noted). MV (10 μgprotein per 0.3×10⁶ cells) were then added for 3-24 hours. In someexperiments, cells were first co-incubated with MV for 3-24 hours, thenwashed and treated further with IRX-2 or cytokines or treated for theindicated time period with MV and IRX-2 added simultaneously. Inselected blocking experiments, anti-Fas neutralizing monoclonalantibody, ZB-4, the pan-caspase inhibitor, Z-VAD-FMK, or the specificAkt-inhibitor or specific inhibitors for caspase-3, caspase-8 andcaspase-9 were added at the indicated concentrations prior to MVco-incubation.

Cell Surface Staining:

MV and/or IRX-2 co-incubated CD8+ Jurkat cells or activatedT-lymphocytes (at least 300,000 cells/tube) were washed twice instaining buffer (0.1% w/v BSA and 0.1% w/v NaN₃). Cells were stained forcell surface markers as previously described. Briefly, cells wereincubated with the optimal dilution of each antibody for 20 minutes atRT in the dark, washed twice with staining buffer and finally fixed in1% (v/v) paraformaldehyde (PFA) in PBS. The following antibodies wereused for surface staining: anti-CD3-ECD, anti-CD4-PE, anti-CD8-PCS,anti-Fas-FITC and anti-FasL-PE.

Flow Cytometry:

Four color flow cytometry was performed using a FACScan flow cytometer(Beckman Coulter) equipped with Expo32 software (Beckman Coulter).Lymphocytes were gated based on morphology, and debris, MV as well asmonocytes and granulocytes were excluded, collecting data on at least10⁵ cells. For the analysis of activated primary T lymphocytes, gateswere restricted to the CD3⁺ CD8⁺ or CD3⁺ CD4⁺ T-cell subsets. Data wasanalyzed using Coulter EXPO 32vl.2 analysis software.

Annexin V Binding Assay:

Annexin V (ANX) binding to MV and/or IRX-2 co-incubated CD8+ Jurkatcells or activated T lymphocytes was measured by flow cytometry toevaluate spontaneous or in vitro induced apoptosis. Followingsurface-staining with antibodies to CD3, CD8 or CD4, the cells wereresuspended in Annexin-binding buffer and incubated with FITC-conjugatedAnnexin V for 15 minutes on ice. Additional staining with7-amino-actinomycin D (7-AAD) was performed to discriminate dead andlive cells. The cells were analyzed by flow cytometry within 30 minutesof staining.

Measurement of Caspase Activation:

Total cellular Caspase activity was tested by intracellular staining ofactivated caspases using a pan caspase inhibitor, CASPACE FITC-VAD-FMKIn Situ Marker (Promega, Madison, Wis.). Cells were resuspended in PBSand FITC-VAD-FMK was added at a final concentration of 5 μM. The cellswere incubated for 20 minutes at 37° C., 5% CO₂ and washed with PBS.Then cells were stained for cell surface receptors, fixed with 1%paraformaldehyde and analyzed by flow cytometry. The specific activationof caspase-3 and caspase-7 was measured using the VYBRANT FAM caspase-3and -7 Assay Kit from Invitrogen (Carlsbad, Calif.) according to themanufacturers' instructions. Briefly, cells were resuspended in PBS andstained with a 150× dilution of the carboxyfluorescein (FAM)-labeledFMK-caspase inhibitor for 60 minutes at 37° C., 5% CO₂. Then the cellswere washed in wash buffer and fixed with 1% paraformaldehyde. The cellswere analyzed by flow cytometry with the fluorescein measured on the FL1channel.

Measurement of the Mitochondrial Membrane Potential:

The loss of mitochondrial membrane potential (MMP) as a hallmark ofapoptosis was measured using the MITOPROBE JC-1 Assay Kit fromInvitrogen (Carlsbad, Calif.). The cationic dye JC-1(5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazolylcarbocyanineiodide) exists in healthy cells as a green monomer in the cytosol andalso accumulates as red aggregates in the mitochondria. In apoptotic andnecrotic cells, JC-1 remains only in the cytoplasm due to mitochondrialdepolarization, which can be detected by flow cytometry as a decrease inthe red/green fluorescence intensity ratio. CD8+ Jurkat cells oractivated T lymphocytes were incubated in PBS containing 2 μM of JC-1for 30 minutes at 37° C., 5% CO₂. An aliquot of the cells was treatedwith 50 μM of the mitochondrial uncoupler carbonyl cyanide3-chlorophenylhydrazone (CCCP) during the staining period as a positivecontrol for mitochondrial depolarization. The cells were analyzed usinga flow cytometer immediately after staining.

Evaluation of Apoptosis-Related Proteins:

Expression of anti-apoptotic proteins Bcl-2, Bcl-xL, FLIP and Mcl-1 andthe pro-apoptotic proteins Bax, Bim and Bid was investigated in CD8+Jurkat cells or activated primary T lymphocytes using multicolor flowcytometry. The cells were first stained for surface T-cell markers asdescribed above. For intracellular staining of apoptosis-relatedproteins the cells were fixed with 1% (v/v) paraformaldehyde in PBS atRT for 10 minutes and then permeabilized with saponin (0.1% v/v in PBS)for 15 minutes at 4° C. Next, the cells were stained for 30 minutes at4° C. with FITC- or PE-conjugated antihuman Bcl-2, Bax and Bcl-xL orunconjugated antibodies for FLIP, Bim, Bid or Mcl-1, followed by a washwith 0.1% saponin. Samples stained with unconjugated antibodies werefurther incubated with a FITC-conjugated goat anti-rabbit IgG for 15minutes at room temperature. After washing with 0.1% saponin, cells werefixed in 1% (v/v) paraformaldehyde. Isotype control matched antibodieswere used for both surface and intracellular controls and all antibodieswere pre-titered on fresh PBMC.

TUNEL Assay:

DNA fragmentation was measured using the In Situ Cell Death DetectionKit, TMR red (Roche, Indianapolis, Ind.). Briefly, cytospin preparations(100,000 cells/slide) of MV- and IRX-2 treated T cells were air-driedand fixed with 4% (v/v) paraformaldehyde (PFA) in PBS for 1 hour at RT.Slides were rinsed with PBS and incubated with permeablization solution(01% Triton X-100 in 0.1% sodium citrate) for 2 minutes on ice. Then theslides were washed twice with PBS and incubated with 20 μl of the TUNELreaction mixture for 1 hour at 37° C. in a humidified chamber in thedark. Then the samples were washed extensively with PBS and incubated ina medium with 4′,6-diamidino-2-phenylindole (DAPI; Vector Laboratories,CA) to trace cell nuclei. Slides were evaluated in a Nikon Eclipse E-800fluorescence microscope under ×200 magnification. For digital imageanalysis, Adobe Photoshop 6.0 was used. A minimum of 300 cells wererandomly counted in a microscopic field to determine the percentage ofcells with DNA fragmentation.

Statistical Analysis:

Statistical analysis was performed using the Student's t-test. P values<0.05 were considered significant.

Example 1 IRX-2 Protects Both Jurkat T Cells and Primary T Lymphocytesfrom Cell Death Mediated by a Variety of Apoptosis-Inducing Agents

To determine whether IRX-2 protects T cells from apoptosis mediated bytumor-derived microvesicles (MV), CD8+ FasL-sensitive Jurkat cells werepre-incubated with a 1:3 dilution of IRX-2 (approximately 4 ng/mL or 90IU/mL IL-2) for 24 hours and subsequently treated them with 10 μg oftumor-derived MV (10 μg), CH-11 (400 ng/mL) or staurosporine (1 μg/mL)for 3 hours. As shown in Applicants' previous studies, the co-incubationof Jurkat cells with MV caused marked apoptosis, demonstrated byenhanced Annexin V binding (FIGS. 4A and 4C) and binding of FITC-VAD-FMKindicative of caspase activation (FIGS. 4B and 4D). Dead cells (7-AAD+)were excluded and the gate was set on 7-AAD negative CD8+ Jurkat cells.Upon pre-incubation of Jurkat T cells with IRX-2, the MV-inducedapoptosis, as detected by both assays, was significantly reduced (FIGS.4A-4D).

Interestingly, it was found that IRX-2 was effective not only againstMV-induced apoptosis, but also provided protection of Jurkat T cellsagainst FasL-induced (CH-11-Ab) and cytotoxic drug-induced(staurosporine) apoptosis. FIGS. 12C-12D shows IRX-2 significantlyreduced apoptosis induced by each of these agents as measured bydecreased Annexin V binding (FIG. 4C) and by reduced caspase activation(FIG. 12D). Results shown in FIG. 4C and FIG. 4D are representative of 3independent experiments (*p <0.05; ** p <0.002).

IRX-2-mediated protection was also observed when using primaryblood-derived CD8+ and CD4+ T lymphocytes. The data in Table 1illustrates the protective effect of IRX-2 on both MV- or CH-11-inducedapoptosis as indicated by decreases in caspase activation in these cells(Table 1). Similar decreases of Annexin V binding were observed withIRX-2 (data not shown). CD8+ T cells showed a significantly greatersensitivity to MV-induced apoptosis than CD4+ T cells, but in both thesesubsets, IRX-2 pre-treatment provided a strong protection againstMV-induced apoptosis, as determined by a total decrease in thepercentage of T cells with caspase-activation. IRX-2 also protected bothcell subsets from CH-11 Ab induced apoptosis (Table 1). Taken together,these findings indicate that IRX-2 effectively protects primary T cellsand cell lines from MV- or anti-Fas CH-11 Ab-induced apoptosis as wellas from intrinsic apoptosis associated with staurosporine-inducedmitochondrial changes. Such results strengthen the argument that IRX-2provides significant protection from several different types ofapoptotic stimuli including that derived from tumors as well intrinsicmechanisms that may be induced by chemotherapy, radiotherapy or viralinfection for example.

TABLE 1 IRX-2 protects activated peripheral blood CD8+ and CD4+ T-lymphocytes from MV- and Fas-induced apoptosis^(a) CD8+ cells CD4+ cellsmean % of mean % of FITC-VAD- FITC-VAD- FMK+ FMK+ cells ± SD p-value^(b)cells ± SD *p-value^(b) untreated cells 14.8 ± 4.8 12.6 ± 2.3 no IRX-2 +CH-11 Ab 52.8 ± 4.9 41.2 ± 9.8 + IRX- + CH-11 Ab 15.0 ± 3.5 0.0010 15.4± 6.1 0.0510 no IRX-2 + MV  68.9 ± 10.4 49.8 ± 8.0 + IRX-2 + MV  26.5 ±10.3 0.0006 13.9 ± 6.4 0.0211 ^(a)Activated CD8+ or CD4+ cells werepre-incubated with IRX-2 for 24 hours (at 1:3 final dilution, contains90 IU/ml IL-2; see Materials and Methods for add'l cytokine conc.details) and then treated with 10 μg MV or CH-11 antibody (Ab) (400ng/mL) for an additional 24 hours. Cells were analyzed for caspaseactivation by FITC-VAD-FMK staining via flow cytometry. Results are meanpercentage ± SD of 3 independent experiments. ^(b)The p values are fordifferences between no IRX-2 and +IRX-2 treated cells.

Example 2 IRX-2-Mediated Protection from Apoptosis is Time- andConcentration-Dependent

To better understand the protective effects of IRX-2 on T cells, CD8+Jurkat cells were pre-incubated with IRX-2 (fixed dilution of 1:3=90IU/ml IL-2) for incrementally longer time periods (0-24 hours) or withincreasing concentrations of IRX-2 (as indicated) for a fixed 24 hourperiod and subsequently treated with MV (10 μg) for 3 hours (FIGS. 5Aand 5B, respectively). Apoptosis was assessed using FITC-VAD-FMKstaining of activated caspases by flow cytometry. IRX-2 was found toblock MV-induced apoptosis, and this inhibition was time-dependent, asextending the time of the pre-incubation with IRX-2 intensified itsprotective effects. A maximal inhibition was observed after 24 hours ofMV treatment (FIG. 5A). Pre-incubation of T cells with different IRX-2concentrations showed a dose-dependent inhibition of apoptosis caused byMV (FIG. 5). At the highest possible concentration (i.e., undilutedIRX-2), IRX-2 completely inhibited the induction of apoptosis by MV(FIG. 5B). Results are mean percentage±SD of 4 independent experiments.The fact that IRX-2-mediated inhibition is both time and concentrationdependent demonstrates pharmacologically that the effects are specificto the drug.

It was also desired to determine whether IRX-2 could protect T cellsfrom apoptotic cell death once the apoptotic cascade had been initiated.To address this question, CD8+ Jurkat cells were untreated, treated withIRX-2 (1:3 dilution) for 24 hours (+IRX), MV for 3 hours (+MV),pre-incubated with IRX-2 for 24 hours and then treated with MV (10 μg)for 3 hours (+IRX→MV) or first incubated with MV and then treated withIRX-2 (+MV→IRX-2) or incubated with both agents simultaneously (+MV andIRX) for 3 hours or 24 hours, respectively. Activation of caspases wasanalyzed by flow cytometry. Results are mean percentage±SD from arepresentative experiment of 3 performed (*p <0.002 compared toMV-treated sample). In comparison to the effects of IRX-2 treatmentprior to the addition of MV, apoptosis was reduced by about 50% aftersimultaneous co-incubation of T cells with MV+IRX-2 (FIG. 6). When IRX-2was added 3 hours after treatment with MV, the protective effect ofIRX-2 was completely abrogated. Since IRX-2 was obviously not able toovercome the apoptotic cascade already initiated by MV, it acts througha protective mechanism rather than through a reversal of ongoingapoptotic processes initiated by MV.

Example 3 Comparison of the Protective Effect of IRX-2 with the Effectof the Survival Cytokines IL-7 and IL-15: Caspase-Activation in JurkatCD8+ Cells after Treatment with Tumor-MV

Jurkat CD8+ cells were plated at a density of 300,000 cells/100 μL/wellin a 96 well-plate and incubated for 24 hours with IRX-2 (1:3 finalconcentration), IL-7 (10 ng/mL), IL-15 (10 ng/mL), or both cytokines (10ng/ML each), respectively. The cells were treated for 3 hours withPCI-13/FasL-MV (15 μg). Jurkat CD8+ cells heated for 10 minutes at 56degrees C. were used as a positive control. The cells were harvested,washed in 1 mL PBS, resuspended in 500 μL PBS, and stained with 5 μMVAD-FITC at 37 degrees C. for 20 minutes. Then the cells were washed inPBS and stained for 15 minutes for CD8-PE-Cy5. After washing, the cellswere fixed in 1% PFA and analyzed by multiparametric flow cytometry.

The percent of activated caspase-VAD-FITC binding CD8+ Jurkat cells weredetermined for each treatment group as shown in FIG. 7. The MV-inducedapoptosis (no IRX-2 lane; 50% cells undergoing apoptosis) wasdramatically inhibited by pre-treatment with either IRX-2 alone (11%apoptotic cells=4.5 fold reduction) or a mixture of IL-7 and IL-15 (5%apoptotic cells=10 fold reduction). Singly, neither IL-7 nor IL-15 alonewas able to reduce the level of apoptosis below the control MV-inducedlevel. IRX-2 does not contain either IL-7 or IL-15 both of which arerecognized as potent “survival” factors of lymphoid cells. Thisdemonstrates that the apoptosis-inhibiting activity of IRX-2 is theresult of a synergistic combination of biologically active componentsand cannot be reproduced by a single recombinant cytokine.

Example 4 Protective Effect of IRX-2 and Survival Cytokines IL-7 andIL-15: Caspase-Activation in Activated CD8+ and CD4+ T-Cells afterTreatment with Apoptosis Inducers

Non-adherent cells from leukocyte units (buffy coats) were thawed,60×10⁶ cells (in 60 mL 10% FCS, RPMI-medium) were activated withCD3/CD28 Dynal beads (1 bead/cell) for 3 days. After activation, cellswere washed and CD8+ and CD4+ cells were isolated by magnetic separation(positive selection, Miltenyi MicroBeads). 300,000 cells were plated in96 well-plates in 100 μL/well and incubated with IRX-2 (1:3) or thecytokines II-7 and IL-15 (100 ng/mL) for 24 hours. The cells were thentreated for an additional 24 hours with PCI-13/FasL-MV (15 μg) (FIGS.8A, 8B) or CH-11 Ab (400 ng/mL) (FIGS. 9A, 9B) to induce apoptosis.

After incubation, the cells were harvested, washed in 1 mL PBS,resuspended in 300 μL PBS and stained with 3 μM VAD-FITC at 37 degreesC. for 20 minutes. The cells were washed in PBS and stained for 15minutes for CD8-PE-Cy5 or CD4-PE-Cy5, as indicated. After washing, thecells were fixed in 1% PFA and analyzed by multiparametric flowcytometry.

In response to apoptosis induction via tumor cell line PCI-13/FasL-MV,the percentage of cells binding caspase-VAD-FITC (indicator of cellswere undergoing apoptosis) was determined for pre-activated humanperipheral blood-derived CD4+ (FIG. 8A) or CD8+ (FIG. 8B) cells that hadbeen pre-incubated with IRX-2 alone or the indicated recombinantcytokines. These data show that IRX-2 is able to inhibit apoptosis asseen previously on Jurkat CD8+ cells (FIG. 4-6) however the degree ofinhibition appears less than what was observed on Jurkat cells. In thisexperimental situation where primary human (non-cell line) T cells areemployed, IL-7 and IL-15 were similarly effective compared to IRX-2,either alone or combined. This apparent difference is most likelyrelated to the use of a heterogeneous T cell population from peripheralblood rather than the cloned homogeneous Jurkat cell line. Nevertheless,in either situation and on both CD4+ and CD8+ populations, IRX-2 didindeed inhibit apoptosis in response to the tumor-derived MV.

In an extension to the above findings, a similar experiment wasundertaken to evaluate percent caspase-VAD-FITC binding of pre-activatedhuman peripheral blood-derived CD4+ (FIG. 9A) or CD8+ (FIG. 9B) cells inthis case treated with an alternative apoptosis inducer, anti-Fasantibody (CH-11). In this context, IRX-2 is the most effective atinhibiting apoptosis induction compared to either IL-7 or IL-15 or bothcombined. This was true for both the CD4+ and CD8+ populations takenfrom normal blood donors. Such results strengthen the argument thatIRX-2 provides significant protection from several different types ofapoptotic stimuli including that derived from tumors.

Example 5 The Survival Signals Promoted by IRX-2 Greater than theProtective Effects of Other Recombinant Survival Cytokines

Since IL-2 is a principal cytokine in IRX-2 (˜90 IU/mL IL-2 at the 1:3dilution used), the observed anti-apoptotic activity of IRX-2 could bein part IL-2-dependent. On the other hand, synergy with other cytokinespresent in IRX-2 could promote survival. Activated CD8+ and CD4+ T-cellswere incubated with either 100 IU/mL of recombinant human IL-2, a doseapproximating that present in the 1:3 IRX-2 dilution, or IRX-2 (˜90IU/mL at 1:3) and compared for the ability to inhibit MV- or CH-11antibody-induced apoptosis. As shown in Table 2A, IL-2 had a similarprotective effect against MV-induced apoptosis as IRX-2 in CD8+ T cells,but had a lower protective effect in CD4+ T cells. In terms ofprotection against CH-11Ab-induced apoptosis, IL-2 was significantlymuch less effective than IRX-2 in enhancing survival of CD4+ T cells andhad almost no effect in CD8+ T cells (Table 2B). These findings indicatethat the survival-enhancing potential of IRX-2 is greater than that ofits main cytokine IL-2 and that support by other cytokines that arepresent in IRX-2 at very low physiological concentrations contribute tothese effects.

It is likely that cytoprotective effects of IL-2 in IRX-2 are enhancedby the presence of IFNγ and GM-CSF, which in combination, could mediateimmuno-potentiating effects. The role of other components of IRX-2(e.g., IL-1α, IL-6, IL-8, TNFα) in promoting T-cell survival is lessclear, although studies have shown that, depending on tissue locationand concentration, some of these pro-inflammatory cytokines can alsosupport anti-tumor immune responses. It is important to note that afunctional synergism amongst the various components of IRX-2 waspreviously described, demonstrating, for example, that IRX-2 was able toinduce maturation of dendritic cells to a greater extent than comparablelevels of TNFα alone.

Additionally, the protective effect of IRX-2 was compared with theactivity of recombinant IL-7 and IL-15, both potent survival cytokinesfor lymphocytes, which are not present in the IRX-2 mixture.Pre-incubation of T cells with these cytokines at a concentration of 10ng/mL, alone or in combination, provided protection from MV-induced orCH-11 Ab-induced apoptosis in all cases, although to different extents(Table 2A and B). IL-7 alone only weakly inhibited both CH-11 Ab- andMV-induced apoptosis in both cells subsets in comparison to IRX-2. IL-15alone was as potent as IRX-2 in protection against MV-induced apoptosisbut provided a weaker survival signal against CH-11 Ab-inducedapoptosis. A combination of both cytokines blocked apoptosis in CD8+ andCD4+ cell subsets, and the level of apoptosis inhibition was similar tothat mediated by IRX-2, but only in case of MV-induced apoptosis (Table2A and B). Thus, the protective effects of IRX-2 were comparable to orin some cases (e.g., protection of CD8+ cells) even stronger than thoseof the recombinant survival cytokines, IL-7 and IL-15 in protecting CD4+cells. IRX-2 was found to be significantly more effective thanrecombinant IL-7 in protecting activated CD4+ and CD8+ T cells from MV-and CH-11 Ab-induced apoptosis and had similar protective effects asIL-15. Among the cytokines tested, IRX-2 had the greatest survivalpotency when CH-11 Ab was used to induce apoptosis, implying protectionagainst receptor-mediated apoptosis. It should be noted that theconcentrations of IL-7 and IL-15 used in these experiments arerelatively high and not physiological levels, again suggesting a strongsynergy between the components of IRX-2.

TABLE 2 Anti-apoptotic effects of IRX-2 in comparison to IL-2, IL-7 andIL-15 in (a) MV-induced or (b) CH-11 Ab-induced^(a) apoptotic primary Tcells. CD8+ cells CD4+ cells mean % mean % of FITC-VAD- of FITC-VAD-FMK+ FMK+ cells ± SD p-value^(b) cells ± SD p-value^(b) A. MV-inducedapoptosis control 21.8 ± 3.9 14.3 ± 5.0 no IRX-2 + MV 63.2 ± 4.5 52.8 ±8.7 + IRX-2 + MV 20.6 ± 0.8 0.0006 10.2 ± 0.4 0.0038 +IL-7 + MV 49.5 ±3.5 0.0004 34.5 ± 1.9 0.0124 + IL-15 + MV 16.8 ± 6.2 0.0044 16.7 ± 2.10.0072 + IL-7/IL-15 + MV 11.8 ± 6.2 0.0036 11.9 ± 0.8 0.0031 + IL-2 + MV21.9 ± 2.7 0.0002 22.2 ± 2.7 0.0113 B. CH-11 Ab-induced apoptosiscontrol 12.3 ± 2.8 11.4 ± 1.3 no IRX-2 + MV 50.0 ± 0.2 46.8 ± 3.0 +IRX-2 + MV 13.4 ± 3.0 0.0007 12.0 ± 1.7 0.0001 +IL-7 + MV 37.7 ± 1.40.0015  31.8 ± 10.6 + IL-15 + MV 28.8 ± 3.1 0.0020 21.1 ± 5.3 0.0086 +IL-7/IL-15 + MV 24.2 ± 4.9 0.0028 20.2 ± 0.1 0.0010 + IL-2 + MV 44.2 ±6.9 31.7 ± 3.0 0.0128 ^(a)Activated primary CD8+ and CD4+ T-cells werepre-incubated with IRX-2 (1:3 dilution, includes ~90 IU/ml IL-2, seeMaterials & Methods.), recombinant human IL-2 (100 IU/mL), IL-7 (10ng/mL), IL-15 (10 ng/mL) or IL-7 and IL-15 (both 10 ng/mL) for 24 hoursand then treated with 10 μg MV or 400 ng/mL CH-11 antibody (Ab) for anadditional 24 hours. Activation f caspases was analyzed by flowcytometry. Data are mean percentages of FITC-VAD-FMK+ cells ± SD.^(b)The p values refer to significant differences between cellspre-treated with IRX-2 compared to MV alone or cells pre-treated withthe cytokine indicated compared to those pre-treatment with IRX-2.

Example 6 IRX-2 Provides Protection Against MV-Induced Apoptosis atVarious Steps in the Apoptotic Pathway

Having shown IRX-2-mediated protection extends to primary T cells aswell as the Jurkat cell line and several inducing agents, we continuedby evaluating the ability of IRX-2 to inhibit downstream steps in theapoptotic process using the co-incubation of Jurkat cells with MV. CD8+Jurkat cells were either untreated, incubated with 10 μg MV for 3 hoursor pre-treated for 24 hours with IRX-2 (1:3 dilution) and then incubatedwith MV for 3 hours. CD8+ Jurkat cells were also co-incubated with MVand 20 μM of the pan-caspase inhibitor Z-VAD-FMK (zVAD) or co-incubatedwith MV and the anti-Fas neutralizing mAb ZB4 (10 μg/mL) (controls).Results are the mean MFI±SD of 3 independent experiments. As shown inFIG. 13A, MV-treatment of Jurkat cells led to a very strong increase inthe mean fluorescent intensity (MFI) of caspase-3/-7-FAM, a dye whichspecifically binds to activated caspase-3 and caspase-7, the maineffector caspases of both receptor- and mitochondrial-mediatedapoptosis. Pre-treatment with IRX-2 completely prevented the MV-inducedinduction of caspase-3 and -7 activity as well as the irreversiblecaspase inhibitor zVAD and the anti-Fas neutralizing monoclonal antibody(mAb) ZB4. Caspase-3 activation by MV was also detected by Westernimmunoblot analysis, where a dramatic decrease in the protein level ofthe inactive caspase-3 pro-form and a simultaneous increase of theactive cleaved form was observed in MV-treated Jurkat T cells over 24hours (FIG. 10B, lanes 3 and 4). IRX-2 pre-treatment effectively blockedinduction of the active cleaved form (FIG. 10B, lanes 5 and 6). Thecells were either untreated, treated with IRX-2 (1:3 dilution) for 24hours (+IRX), treated with MV (10 μg) for 3 hours (+MV 3 hours) or 24hours (+MV 24 hours) or pre-incubated with IRX-2 for 24 hours and thentreated with MV (10 μg) for 3 hours or 24 hours (+IRX→MV). Whole celllysates of the cells were separated on SDS-PAGE and transferred to PVDFmembranes for subsequent Western blotting. Activation of caspase-3 isshown as a decrease in the inactive pro-form and the appearance of theactive subunits p17 and p10. Results are representative of 3 Westernblots.

In addition, IRX-2 blocked the MV-induced loss of mitochondrial membranepotential (MMP) in Jurkat T cells (FIGS. 11A and 11B). This block wascomparable to that provided by the two inhibitors z-VAD and ZB4 (FIG.11B). CD8+ Jurkat cells were analyzed by flow cytometry for a decreasein red fluorescence of the cationic dye JC-1, indicating a loss of MMP.Percentage of JC-1 red-negative cells were determined in cultures ofCD8+ Jurkat cells after no treatment, 24 hours treatment with MV (10 μg)alone (no IRX) or pre-treated for 24 hours with IRX-2 (1:3 dilution) orMV in combination with the apoptosis-inhibitor Z-VAD-FMK (pan-caspaseinhibitor; conc) or ZB4 (anti-Fas neutralizing mAb, conc/dose). Cellstreated with 50 μM carbonyl cyanide 3-chlorophenylhydrazone (CCCP), aprotonophore that dissipates the H+ gradient across the innermitochondrial membrane, were used as a positive control. Results aremean±SD of 3 independent experiments (*p <0.005; **p <0.002 compared tosample without IRX-2).

Finally, IRX-2 pre-treatment significantly reduced the MV-inducednuclear DNA fragmentation as detected by TUNEL assay (FIGS. 12A and12B), representing the final step in the apoptotic process (p <0.0002;FIG. 12B). CD8+ Jurkat cells were either untreated (a), incubated for 24hours with IRX-2 alone (b) or MV alone for 24 hours (c) or pre-incubatedwith IRX-2 for 24 hours and subsequently treated with MV for 24 hours(d) and then stained by the TUNEL method to reveal DNA strand breaks(red nuclei) indicative of apoptosis. A minimum of 300 CD8+ Jurkat cellswere counted for each treatment group. Results are expressed as the meanpercentage±SD of two independent experiments (*p <0.0002 compared toMV-treated sample). This data therefore confirms that IRX-2 exhibitsprotective effects at each of the relevant steps that culminate in Tcell death.

Example 7 IRX-2 Protects T Cells from MV-Induced Down-Regulation of JAK3and STAT5 Expression

It has been previously observed that MV derived from sera of patientswith cancer down-regulate expression of molecules mediating the common γchain cytokine receptor signaling pathway, including JAK3 and STAT5.Since this pathway is essential for the development, maintenance andsurvival of lymphocytes, and in particular, of CD8+ cells, effects of MVand IRX-2 on JAK3 and STAT5 expression in CD8+ Jurkat cells were nextexamined.

CD8+ Jurkat cells were untreated or treated with IRX-2 (1:3 dilution)and MV in different combinations. Whole cell lysates of cells from eachtreatment group were separated on SDS-PAGE and transferred to PVDFmembranes for subsequent Western blotting. The expression levels ofJAK3, phosphorylated and total STAT5, CD3 and FLIP were analyzed byprobing membrane with specific antibodies. Reprobing with β-actinantibody confirmed equal protein loading.

The results shown are representative of 4 experiments performed. Aspreviously observed, MV caused a significant down-regulation of JAK3 inT cells (FIG. 18, panel 1, compare lanes 1 and 3), which intensifiedwith the extended time of co-incubation (FIG. 18, panel 1, lanes 3 and4). While IRX-2 alone did not increase JAK3 expression (FIG. 18, panel1, compare lanes 1 and 2), it was able to completely reverse theMV-induced JAK3 down-regulation and restore its expression (FIG. 18,panel 1, compare lanes 3, 4 with lanes 5, 6). Furthermore, IRX-2 causeda strong activation of STAT5, a JAK3 signal transducer, as indicated byphosphorylation of this protein (FIG. 18, panel 2, lane 5). Thisdramatic activation of STAT5 was sustained even after prolonged (24hours) incubation with MV (FIG. 18, panel 2, lane 6). Additionally, aloss in CD3-ζ expression in T cells was observed after MV treatment.Here again, pre-incubation with IRX-2 protected T lymphocytes fromMV-mediated CD3-ζ down-regulation (FIG. 18, panel 4, compare lanes 3, 4with lanes 5, 6).

These changes are all consistent with IRX-2 mediating protection fromapoptosis via the cytokines present in IRX-2, especially via the primarycytokine IL-2 which is known to signal through the IL-2 receptor and thedownstream intracellular signaling molecules Jak3/Stat5. These dataelucidate downstream molecular targets of IRX-2 that are central insending survival and stimulation signals in lymphoid cells.

Example 8 IRX-2 Reverses the MV-Induced Imbalance of Pro- andAnti-Apoptotic Proteins

To further examine the mechanisms through which IRX-2 promotedprotection of T cells from apoptosis, expression levels of various pro-and anti-apoptotic proteins were measured in activated, MV-treated Tlymphocytes and CD8+ Jurkat cells in the presence or absence of IRX-2 byquantitative flow cytometry. Table 3 shows expression levels of severalapoptosis-related proteins as mean fluorescence intensity (MFI) inactivated CD8+ cells before and after MV treatment. Incubation of Tcells with MV caused a significant up-regulation of the pro-apoptoticproteins Bax and Bim, and a concurrent down-regulation of anti-apoptoticBcl-2, Bcl-xL, FLIP and Mcl-1 (Table 3A). This is consistent withApplicants' previous findings indicating that MV induce apoptosis of Tcells. While absolute protein levels are important, it is the ratio ofpro-/anti-apoptotic protein levels present in the cell that actuallydetermines cell fate. Thus, changes in these ratios are much moreinformative of cell state (Table 3B). Dramatic changes of the Bax/Bcl-2,Bax/Bcl-xL and Bim/Mcl-1 ratios upon treatments with MV or IRX-2+ MVwere observed. A significant pro-apoptotic shift in these ratiosoccurred in CD8+ cells upon incubation with MV. In contrast,pre-treatment of T cells with IRX-2 caused a dramatic decrease in theseratios rendering them congruent with those present in untreated cells.(Table 3B), as shown in FIG. 14A. Activated peripheral blood (PB) CD8+cells were pre-incubated with IRX-2 (at 1:3 dilution) for 24 h and thentreated with 10 μg MV for additional 24 h. Expression levels (meanfluorescence intensity) of different pro- and anti-apoptotic proteinwere measured by quantitative flow cytometry. As shown in FIG. 14B IRX-2treatment is able to maintain levels of the anti-apoptotic proteinsBcl-2 and Mcl-1 after MV treatment and down regulates expression of thepro-apoptotic protein Bax.MV and IRX-2 had little or no effect on theexpression of pro-apoptotic FasL and Bid (data not shown). Similarresults were obtained after IRX-2 incubation and MV treatment ofactivated primary CD4+ cells and CD8+ Jurkat cells.

The balance of pro-versus anti-apoptotic proteins determines whether thecell will complete the apoptotic process resulting in death of the cell.IRX-2 reverses the MV-induced shift toward apoptosis leading toprotection from apoptosis. The fact that IRX-2 works to up-regulate coreanti-apoptotic proteins such as BCL-2, demonstrates that it is a generalinhibitor of apoptosis in lymphoid cells and is beneficial in protectingthese cells from a wide variety of tumor derived factors.

TABLE 3 MV and IRX-2 modulate the expression of pro- and anti-apoptoticproteins. The mean fluorescence intensity ± SD (a) and ratios (b) ofpro- and anti-apoptotic proteins of MV- and IRX-2-treated activated CD8+cells^(a) are indicated below. A. + MV + IRX-2 + MV untreated mean pvalue mean p value mean fluorescence (compared to fluorescence (comparedto fluorescence intensity ± untreated intensity ± MV-treated intensity ±SD SD sample) SD sample) Bcl-2  7.7 ± 0.4  1.9 ± 0.1 0.0008  4.8 ± 0.60.0049 Bax 17.9 ± 1.2 40.0 ± 1.5 0.0001 26.8 ± 2.3 0.0003 Bcl-xL 20.1 ±0.1  8.2 ± 0.4 0.0001 11.0 ± 0.8 0.0019 FLIP 42.4 ± 0.6 17.5 ± 0.60.0002 25.5 ± 2.0 0.0030 Bim  8.1 ± 0.3 16.7 ± 1.6 0.0016  9.2 ± 0.30.0020 Mcl-1 37.5 ± 3.8  7.1 ± 1.1 0.0004 35.1 ± 1.6 0.0003 B. Bax/Bcl-2ratio Bax/Bcl-xL ratio Bim/Mcl-1 ratio mean ± SD p value^(b) mean ± SD pvalue^(b) mean ± SD p value^(b) untreated  2.3 ± 0.6 0.89 ± 0.3 0.22 ±0.3 +MV 18.6 ± 1.2 0.0004 4.32 ± 0.5 0.0001 2.35 ± 0.2 0.0011 + IRX-2 +MV 5.58 ± 0.9 0.0002 2.44 ± 0.4 0.0003 0.26 ± 0.1 0.0009 ^(a)Activatedperipheral blood (PB) CD8+ cells were pre-incubated with IRX-2 (at 1:3dilution; containing ~4 ng/ml or 90 IU/ml IL-2) for 24 hours and thentreated with 10 μg MV for additional 24 hours. Expression levels (meanfluorescence intensity) of different pro- and anti-apoptotic proteinwere measured by quantitative flow cytometry. The data are means ± SDobtained in 3 different experiments. ^(b)The p values indicatesignificant changes in ratios between untreated and MV-treated orIRX-2 + MV-treated cells.

Example 9 The Akt/PI3K-Pathway is the Main Downstream Target ofAnti-Apoptotic Activity of IRX-2

The Akt/PI3K signaling pathway is recognized as one of the most criticalpathways in regulating cell survival. Since our findings showed asubstantial influence of IRX-2 on several key proteins of theBcl-2-family, which could be regulated by Akt/PKB, we measured theactivation of Akt-1/2 in response to MV and/or IRX-2 using an antibodyspecific for one of the two major regulatory phosphorylation sites,phosphoserine 473. CD8+ Jurkat cells were untreated or treated withIRX-2 and MV (10 μg) in different combinations as indicated. Whole celllysates of cells from each treatment group were separated on SDS-PAGEand transferred to PVDF membranes for subsequent Western blotting. Theactivation of Akt-1/-2 was analyzed by immunoblotting withSer473-specific anti-phospho Akt mAb. Reprobing with a total-Aktantibody confirmed equal protein loading. Results shown arerepresentative from one experiment out of 3 performed.

In untreated CD8+ Jurkat cells (control) Akt-1/2 was constitutivelyphosphorylated to a level characteristic of Jurkat cells (FIG. 20A,panel 1, lane 1). Pre-incubation with IRX-2 did not enhance basalAkt-phosphorylation (FIG. 15A, panel 1, lane 2). However, when the cellswere treated with MV, a dramatic, time-dependent dephosphorylation ofAkt-1/2 was observed (FIG. 20A, panel 1, lanes 3 and 4). A time-coursestudy with 10 μg of MV showed that Akt dephosphorylation started at 3hours of incubation and intensified over time (data not shown).Pre-treatment of Jurkat cells with IRX-2 completely inhibited MV-induceddephosphorylation of Akt-1/2 at both 3 and 24 hours of treatment (FIG.215A, panel 1, lanes 5 and 6).

This pronounced pro-survival effect of IRX-2 on CD8+ Jurkat cells, whichclearly counteracted the MV-induced Akt inactivation, indicated that Aktmight serve as the main downstream target of IRX-2 signaling. To confirmthis hypothesis, CD8+ Jurkat cells were pre-incubated prior to IRX-2 andMV treatment with a small molecule inhibitor specific for Akt, Akti-1/2,and measured the levels of T-cell apoptosis. CD8+ Jurkat cells werepre-incubated with IRX-2 for 24 h or left untreated. Then cells weretreated with an Akt inhibitor, Akti-1/2 at different concentrations (0-5μM) for 1 hour prior to the addition of MV for additional 3 hours. Thelevel of apoptosis was measured by FITC-VAD-FMK staining and flowcytometry analysis. Results are mean percentage±SD obtained in 3individual experiments (*p <0.05; **p <0.01 compared to MV-treatedsample without IRX-2 and Akt inhibitor).

As shown in FIG. 15B, pre-treatment of the cells with the Akt inhibitorresulted in a gradual abrogation of the anti-apoptotic effect of IRX-2.At a relatively low inhibitor-concentration of 1 μM, the protection fromapoptosis provided by IRX-2 was only slightly inhibited. However, it wascompletely blocked at the inhibitor concentration of 5 μM. At theseinhibitor concentrations, cell viability was not affected (data notshown). This finding shows that Akt is the main downstream coordinatorof the survival signal provided by IRX-2.

Conclusions of Examples 1-9

Confirming previous findings of the Applicants, it was initially showedthat incubation of CD8+ Jurkat cells or activated T lymphocytes with MVinduced a significant level of apoptosis. Tumor-derived MV expressing amembrane form of FasL were purified from supernatants of the PCI-13tumor cell line and co-incubated with CD8+ Jurkat cells or activatedperipheral blood (PB) T cells. FasL, the Fas ligand, is a type IItransmembrane protein belonging to the tumor necrosis factor (TNF)family. FasL-receptor interactions play an important role in theregulation of the immune system and the progression of cancer. Apoptosisis induced upon binding and trimerization of FasL with its receptor(FasR), which spans the membrane of a cell targeted for death. FasL+MVinduced not only the extrinsic receptor-mediated apoptotic pathway, butalso the intrinsic mitochondrial pathway in activated T cells, withaccompanying up-regulation of the pro-apoptotic Bcl-2 family members,Bax and Bim. Pre-incubation of CD8+ Jurkat or activated primary T cellswith IRX-2 suppressed both apoptotic pathways in a dose- andtime-dependent manner. Further, the pre-treatment of T cells with IRX-2provided protection not only against MV-induced cell death, but alsoagainst CH-11 Ab- and staurosporine-induced apoptosis. Since the formerinduces apoptosis mainly through the death receptor pathway and thelatter activates only the mitochondrial pathway, these findings furthershow that IRX-2 can protect T-cells from activation of both theextrinsic and the intrinsic death pathways.

Example 10

The selection of the dose and schedule for the IRX-2 regimen to be usedin experiments was based on studies conducted by IRX Therapeutics. TheIRX Therapeutics study was performed in mice immunized with prostatespecific membrane antigen (PSMA) peptide conjugate and assessed asincrease in footpad swelling. FIG. 21 shows these data and thecharacteristic “bell-shaped” curve.

The study was performed in four groups of patients, as shown in Table 4below. The graph of tumor lymphocyte infiltration and survival for thesegroups are presented in FIGS. 17 and 18, respectively.

TABLE 4 Cumulative Dose of IRX-2 Injections/ Dose of Regimen N injection(Units) day # days IRX-2 (Units) 1 4  ~38 U 1 10   380 U 2 15 ~115 U 110 1,150 U 3 10 ~115 U 2 20 4,600 U 4 6 ~660 U 2 20 26,400 U 

In this study, maximum lymphoid infiltration was achieved for patientstreated with the 10 days of 115 U IL-2 equivalence/day. Survival waspoor in the four patients who received the lowest dose (regimen 1).Similarly, poorer survival was noted in six patients treated with thehighest dose. While survival appeared to be comparable for regimens 2and 3, regimen 2 patients experienced the most significant histologicalresponse as measured by lymphoid infiltration.

The dose of IRX-2 to be studied further was subsequently selected asintermediate between the two most active doses investigated (regimens 2and 3), a dose clearly adequate to achieve significant histologicalchanges in tumor and lymph nodes. Based upon the additionalinconvenience of 20 versus 10 days of treatment and the lesser lymphoidinfiltration in the patients who received the higher IRX-2 dose, a10-day injection protocol with bilateral injection (approximately 2300 Utotal of IRX-2) was selected for the further studies discussed below.

Example 11

A study of the IRX-2 protocol was performed in H&NSCC patients prior tosurgery and/or radiotherapy and/or chemoradiotherapy as described inFIG. 1. IRX-2 was administered bilaterally at 115 Units/site. Twentyseven patients were treated; their demographics summarized in Table 5.

TABLE 5 Number of treated patients 32 Median age (range) 66 (34-86) M:Fratio 25:7 KPS range 70-100 Patient Characteristics Oral 15 Larynx 13Other  4 Stage at Diagnosis I  1 II  5 III 10 IV 15 NA  1 No. (%) Stageof primary tumor T1  1 (4) T2 15 (56) T3  6 (22) T4  5 (19) TX  0 Nodalstage N0  5 (19) N1  8 (30) N2 14 (52) N3  0 NX  0

Radiological studies (CT or MRI) were performed at the onset and priorto surgery and reviewed centrally (Perceptive, Waltham, Mass.). Bloodwas analyzed centrally (Immunosite, Pittsburgh, Pa.) at onset and priorto surgery for various leukocyte populations (Table 6 and 7). Surgicalsamples were sent to a central reference laboratory (Phenopath, Seattle,Wash.) for evaluation of the histological changes and performance ofimmunohistochemistry for various leukocyte markers (Table 8).Appropriate laboratory and clinical measurements were performed toassess toxicology and symptomatic improvement throughout disease-freeand overall survival continue to be monitored.

Clinical Results:

Three patients had objective tumor responses (2PR; 1MR). Four patientsshowed radiological responses (>12.5% reduction); five patients (N2, N2,N1, N1, N1) were down-staged as nodes detected as tumor-positive at thesites and centrally were shown to be negative in the surgical specimens.Four tumors softened (a positive sign), 14 patients had symptomaticimprovement/reduced pain and tenderness, improved swallowing, and lessbleeding. Treatment related side effects were generally mild (grade I orII) and infrequent including nausea, vomiting, dry mouth, constipation,injection site pain, headache, myalgia, anemia, and contusion. A singleexample of dyspepsia grade III was observed. Disease-free andoverall-survival are being followed. Most patients have cleared one yearand survival curves closely parallel those previously observed byApplicants in studies at the National Cancer Institute of Mexico andappear better than case-matched U.S. and Mexican controls.

Example 12

Heparinized blood was collected for immunophenotyping studies todetermine numbers of immune cell subsets including B, T, NK, and Tnaïve, T memory, and T effector cells. Fluorescently tagged monoclonalantibodies to the indicated cell surface markers (or correspondingisotope control) were used to stain fresh, unfractionated whole blood.

The stained and fixed samples were then acquired and analyzed bymulti-parameter flow cytometry using a Beckman Coulter FC500 flowcytometer and CXP TM analysis software. Enumeration of absolute Tlymphocyte subsets using this single platform (flow cytometry only)method that employs Flow Count TM beads has been demonstrated to be moreaccurate than dual (hematology instruments and flow cytometry) platformtechniques (Reimann et al., 2000). Table 6 below presents a list of theimmune markers analyzed by ImmunoSite and their role in an immunization.

TABLE 6 Immune Markers Analyzed & Role in Immune Response Cell MarkerRole T cell CD3 Mediates cellular immunity B cell CD3− CD19+ CD14−Mediates humoral immunity Helper T cell CD3+ CD4 Makes cytokines,provides B cells “help” Cytotoxic T cell CD3+ CD8 Kills tumor cellsNaive T cell (T_(N)) CD3+ CD45RA+ Antigen naive or very early CCR7+post-primary stimulation; lymph node homing ability Central Memory CD3+CD45RA− Long-lived memory cell, T cell (T_(CM)) CCR7+ low effectorfunction; homes to lymph nodes Effector Memory CD3+ CD45RA− Intermediateeffector T cell (T_(EM)) CCR7− function; shorter half-life in vivo;seeds tissues/tumors over lymph nodes Effector T cell CD3+ CD45RA+Highest effector function (T_(EMRA)) CCR7− (e.g. cytolysis); localizesbest to tissues/tumor

For the purposes of the present invention, only the cell populationsdirectly relevant to evaluating the hypothesis of whether animmunization occurred or not are discussed herein.

The developmental pathways for T lymphocytes, especially CD8+ T cells,have been intensively studied over the last decade with a particularfocus on CD8+ T cells since they are most closely associated witheffective anti-tumor immunity. Both CD4+ helper T cells and CD8+cytotoxic T cells can be subdivided into reciprocal CD45RA+ and CD45RO+subpopulations. CD45RA+ cells have previously been termed naïve T cells;however, more recent work indicates that these T cells in blood comprisenaïve T cells as well as more fully differentiated effectors oftentermed T_(EMRA) (Lanzavecchia, 2005; Kaech, 2002). CD45RO+ (CD45RA−)memory T cells can also be subdivided into T central memory (T_(CM)) andT effector memory (T_(EM)). These sub-classifications are based uponsurface expression of additional markers including CCR7 (Sallusto, 1999;Tomiyama, 2004). The developmental pathways of these various T cellsubsets and their lineage relationships remain complex. The data andtests for significance are presented in Table 7 below.

TABLE 7 Summary of Immunology Assessments & Tests of SignificanceBaseline Mean to Day Degrees Cell cells/ 21 Std of T P population N mL³Std Dev Difference Dev Freedom value value Baseline Lymphocyte 25 1177.5442.4 −69.6 260.7 24 −1.33 0.1946 Gate B cell 18 275.4 132.2 −74.3 74.817 −4.22 0.0006 Helper T cell 25 817.0 330.7 −65.4 184.0 24 −1.78 0.0884Cytotoxic T 25 351.9 193.3 −4.4 87.9 24 −0.25 0.8061 cell Naïve T cell25 55.6 89.8 −38.2 76.9 24 −2.49 0.0203 Central 25 56.9 84.5 −22.8 48.624 −2.34 0.0280 Memory T cell Effector 25 689.0 354.7 41.2 223.4 24 0.920.3651 Memory T cell Effector 25 395.0 250.2 −35.2 132.7 24 −1.33 0.1968Memory RA T cell

Consistent with the hypothesis that IRX-2 acts on both T cells and DC'sto foster activation, maturation, and enhance endogenous tumor antigenpresentation to naïve T cells, it was observed that the naïve T cellpopulation (CD3+ CD45RA+ CCR7+) decreased between baseline and Day 21.Naïve T cells are initially activated by recognition of antigen whenpresented on the appropriate major histocompatibility complex (MCH)molecules by mature DC's. The subsequent steps of generating T cellmemory and full effector function are not perfectly defined, but it isclear that different subpopulations of T cells as defined by severalmarkers, i.e. CD45RA/RO and CCR7 have distinct functional properties.For example, CCR7 expression confers the ability of the T cell to hometo lymph nodes where the most effective anti-tumor priming occurs.

A significant decline was observed in the naïve T cell population (CD3+CD45RA+ CCR7+) with population levels of 55.6 cells/mL³ at baselinefalling to 17.4 cells/mL³ at Day 21 (p=0.02). A loss of naïve T cellsresults from those cells finding and being stimulated by theirrespective cognate antigen and the differentiating into an alternativefunctional population, either of the two memory or full effectorpopulations.

In addition, the central memory T cell population (CD3+ CD45RA− CCR7+)with the CCR7+ conferred lymph node homing propensity, fell from 56.9cells/mL³ at baseline to 34.1 cells/mL³ at Day 21 (p=0.028). This too isan indicator that immunization to tumor antigens is taking place inresponse to IRX-2 therapy. Studies show that the T_(CM) population of Tcells represents the earlier, more “stem-like” memory population thatupon re-stimulation, preferentially homes to the lymph node where it cangain more effector, e.g. cytolytic function. The significant declineseen in this population is consistent with these T_(CM) cells exitingthe bloodstream and migrating to the draining lymph nodes where theywill be further activated.

After an immunization, one would expect other immune cells to beenlisted in the attack on the antigen-bearing offender. Further supportto the immunization hypothesis was observed in that a significant drop(p <0.01) in B cells was observed. B cells are recruited into lymphnodes where they are exposed to antigen and then exit to be found in thetumor where they presumably produce antibodies capable of attacking thetumor directly or supporting antibody-dependent cellular cytotoxicity(ADCC).

The statistically significant changes and trends observed hereinstrongly show that an immunization of naïve T cells is occurring due toIRX-2 administration. As no other primary interventions were observed inthese patients, it is unlikely that these changes occurred at random.

The hypothesis that IRX-2 treatment induces immunization to autologoustumor antigens is also supported by Applicants' published information onH&NSCC lymph node response following IRX-2 treatment as compared tonon-randomized normal and H&NSCC control patients (Meneses, 2003). Thesalient lymph node response features associated with IRX-2 treatmentwere nodal replenishment and lymphocyte expansion, particularly Tlymphocytes, which were shown to be depleted in the lymph nodes ofuntreated H&NSCC patients (Verastegui, 2002). Nodal expansion thatoccurs during an immunization presumably due to IRX-2 was also observedto be associated with a reversal of sinus histiocytosis, an apparentdendritic cell functional defect. These changes are consistent with animmunization. A prior study confirms that immunization to tumor antigenoccurs at the level of the regional lymph node, not the tumor itself(Maass, 1995).

Histology

When an immunization occurs in lymph nodes, the new killer memory Tcells are thought to develop and then exit the nodes through bloodvessels, and flow into tissues to patrol for the antigenic target (i.e.the immune target). If the antigenic target is identified, the killermemory T cell will infiltrate the tissue to kill the target. When acellular immune response is initiated, other immune cells are recruitedto participate in the kill and clean-up process.

T lymphocyte infiltration into tumors, particularly of CD45RO+ CD8+ Tcells, is evidence of an immunization to tumor antigens and that suchinfiltration correlates with improved survival in a variety of cancersincluding H&NSCC, melanoma, colorectal, and ovarian (Wolf, 1986; Pages,2005; Galon, 2006.

It was hypothesized herein that an IRX-2 induced immunization in lymphnodes would result in lymphocytic infiltrate in the tumor and tumordisruption and the presence of specific immune cells in the tumor wouldprovide evidence of an anti-tumor immune response. It was alsohypothesized that an immune response to the tumor would be evidenced bydiffuse lymphocytic infiltrate, spanning the tumor's peripheral area toits intratumoral area.

Formalin fixed paraffin embedded blocks or unstained slides from primarytumor biopsy and resection specimens were submitted by the clinicalsites to PhenoPath Laboratories (Seattle, Wash.) for hematoxylin andeosin (“H&E”) and immunohistochemistry staining (“IHC”). Paired samplesfrom 26 IRX-2 study subjects were submitted, 25 were evaluable, and onesurgical specimen had no histological evidence of tumor. Two ad-hoccomparator groups of surgical specimens were collected at the end of thestudy for H&E comparison: 25 surgical specimens from MD Anderson, and 10surgical specimens from Stony Brook Health Sciences Center, randomlyselected from untreated H&NSCC surgical specimens.

Immunohistochemistry staining was performed only on the IRX-2 treatedsamples to determine the presence of immune markers in the tumor. Theirmarkers are listed in Table 8.

TABLE 8 Immune Markers Analyzed by IHC Cell Marker Role in ImmuneResponse T cell CD3 Mediate cellular immunity B cell CD20 Produceantibody Helper T cell CD4 Make cytokines; help B cells Cytotoxic T cellCD8 Kill tumor Plasma cell CD138 Produce antibody Macrophage CD68 AssistT cell and kill tumor Naive/Effector T cell CD45RA+ Naïve/Effector Tcell Memory T cell CD45RO (RA−) Antigen committed T cell

The presence of IHC stained markers was evaluated under low power andgraded using a prospectively defined 0-100 mm visual analog scale (VAS),where 0 represented 0% presence and 100 represented 100% of cellsstaining positive for the marker. The peroxidase reaction used tohighlight the marker overestimates the area or density of lymphocyteinfiltration as compared to H&E staining, thus making IHC-based densitydeterminations unreliable, but IHC remains useful for elucidating therelative relationships between and among cell types.

H&S Studies: Methods and Analyses

Three analyses were performed comparing the H&E stained slides. Twoanalyses were blinded feature extractions from the 25 IRX-2 treated and25 untreated surgical specimens from MD Anderson, one for tumor featuresand one for immune response features. The third analysis was anidentical but unblended immune response feature extraction from the 10H&E stained slides from Stony Brook. In each case, features wereextracted and quantified using a VAS on case report forms.

Two assessments were made for each of the immune response features, thefirst assessment was the overall presence of the marker across theentire surgical specimen and the second was to the degree to which thelocation of the infiltrate was peripheral or intratumoral.

An overall assessment was made taking into account: lymphocyteinfiltration, its density, its balance between tumor and infiltration,and other features that comprise the gestalt impression of the tumor.The other sub-features include the extent of fibrosis and necrosis,suggesting where tumor was but is no longer and in the case of welldifferentiated squamous cell cancer, a concentration of keratin pearlswith minimal or not tumor surrounding it is another sign of tumordestruction. An “Active Immunologic Response” includes lymphoidinfiltration evidence of damage created by the immune system, and thedegree to which tumor is no longer viable and disrupted—in short theextent and process by which the host is combating the tumor. An exampleof the lymphocyte infiltration sub-feature of the “Active ImmuneResponse” is presented in FIGS. 19 and 20.

One of the dominant sub-features on the Active Immune Response variableis the localization and intensity of the lymphocyte infiltration (LI)that are observed in patients treated with IRX-2. Surgical specimensdemonstrating this reaction in both IRX-2-treated patients and thead-hoc comparator groups demonstrated marked increases in the density ofoverall LI, peritumoral LI, and intratumoral LI.

Based on the pre-specified critical point of 50 mm or greater on theVAS, the analysis showed different Active Immunologic Response ratesamong the three groups of surgical specimens as showed in Table 9 below.

TABLE 9 Active Immune Group Patient w/AIR Total Patients ResponseRate 1. IRX-2 Treated 11 25 44.0% 2. MD Anderson 6 1 24.0% 3. StonyBrook 1 10 10.0%

The increase in the frequency of those patients demonstrating an ActiveImmune Response went from 20% in the pooled MD Anderson and Stony Brookgroups to 44% in the IRX-2 treated group (p <0.05 by Chi square test).

Determination of Peritumoral vs. Intratumoral LI

The location of immune cells in the tumor was also evaluated. It washypothesized herein that an active anti-tumor immune response wouldinclude lymphycytic infiltrate that expanded from the peripheral area toinclude the intratumoral area.

Based upon the VAS analysis for Active Immune Response in theIRX-treated patients, 11 showed intense reactions (≧50, termedresponders) and 14 showed less intense reactions (<50, termednon-responders). A comparison of the LI of these two groups is shown inFIGS. 21A and 21B.

As can be seen, the responders showed a marked increase in LI (both areaand density) of the typical section and compared to the non-responders,the increase in intratumoral LI is proportionally much greater than theperitumoral change.

Immunohistochemistry for the location of various markers helps clarifywhich cells dominate in each region. FIG. 22 shows these results. Theperitumoral infiltrate, representing approximately 25% of the LI in thespecimen was dominated by CD45RA+, CD3+, CD4+ T lymphocytes and CD20+ Blymphocytes. Whereas the intratumoral infiltrate, representingapproximately 75% of the LI in the specimen, was dominated by CD45RO+,CD3+ and CD8+ lymphocytes (i.e. the “killer” effector T cell phenotype)and CD68+ macrophages. FIG. 23 provides a pictoral example of IHCstaining fro CD45RO+ memory T cells in an IRX-2 treated surgicalspecimen. TABLE 10 shows the results of each cell population's presencein the tumor.

TABLE 10 Presence in the Tumor* Cell Population N Overall MeanIntratumoral Mean T cell (CD3) 24 52.3 76.5 B cell (CD20) 24 11.0 21.2Helper T cell (CD4) 24 15.5 53.1 Cytotoxic T cell (CD8) 24 37.8 85.7Macrophage (CD68) 24 42.1 91.5 Effector T cell (CD45 RA) 24 7.4 18.4Memory T cell (CD45 RO) 24 65.4 87.3 *Measurements based on 100 mmVisual Analog Scale (VAS) assessments

The strongest support for this immunization hypothesis derives from theexamination of lymphocyte infiltration for infiltration in and aroundthe tumor and the picture of tumor rejection indicating necrosis,fibrosis, and reduced tumor. The rejection patterns are characteristicfor both humoral and cellular immunity with increased B lymphocytes andactivated macrophages within the tumor, respectively. By shifting thebalance back to immunosurveillance by overcoming the immune suppressionseen in cancer patients and restoring immune function, IRX-2 therapycauses the host to reject the tumor and immunize itself against thetumor leading to reduced recurrence and increased survival.

Example 13

In one patient, fused FDG PET/CT scans were compared at day 0 and day21, as shown in FIG. 24. Total glycolitic activity and volume weremeasured and are shown in Table 11.

TABLE 11 Baseline Day 21 % Change Total Glycolytic Activity Tumor 68.9131.36 −54.49% Node 1 72.54 4.97 −93.15% Node 2 14.35 3.15 −78.05% 155.8039.48 −74.66% Volume Tumor 12.16 7.33 −39.72% Node 1 9.46 1.44 −84.78%Node 2 2.28 1.24 −45.61% 23.90 10.01 −58.12%

Example 14

Overall survival was determined both for patients overall (FIG. 25) andfor patients in Stage IVa (FIG. 26). FIG. 25 shows three Kaplan Meirplots of overall survival. The top line is the recently completedmulticenter Phase 2 study (median follow-up of 18.6 months), the middleline is the single center Phase 1/2 study completed 10 years ago and ascompared to the best available comparator-the randomized site matchedRTOG 9501 trial. In both IRX-2 treated groups, survival is above theanatomic site-matched RTOG 9501 trial data. The data suggests that theIRX-2 driven immunization is durable and leads to improved survival.FIG. 26 shows the three Kaplan Meier plots of overall survival the StageIva cohort. The top line is the recently completed multicenter Phase 2study (median follow-up of 18.6 months), the middle line is the singlecenter Phase 1/2 study completed 10 years ago and as compared to thebest available comparator-the randomized site matched RTOG 9501 trial.In both IRX-2 treated groups, survival is above the anatomicsite-matched RTOG 9501 trial data. The data suggests that the IRX-2driven immunization is durable and leads to improved survival in StageIVa patients.

Example 15

IRX-2 was shown to increase regional lymph node size, T cell area anddensity, and reverse sinus histiocytosis. Controls, H&NSCC controls, andH&NSCC patients administered IRX-2 are compared in FIGS. 27A-D. Twentypatients from a total of 50 with H&N SCC treated with the IRX-2 protocolwere selected as having uninvolved regional lymph nodes suitable forevaluation. All displayed clinical responses, either partial responses(PR, >50% tumor reduction) or minor responses (MR, <50%>25% tumorreduction). Complete responders (3/50) and non-responders (5/50) wereexcluded for obvious reasons. Ten untreated H&N SCC control LN specimenswere selected randomly as one control group (H&N SCC control) and 10non-cancer control LN biopsies were selected randomly (control group).Obvious LN pathologies were excluded from the control group. Overall,95% of the IRX-2 LN, 80% of the noncancer controls, and 60% of the H&NSCC controls were adjudged to be stimulated Overall, the LN ofIRX-2-treated patients showed a high percentage of stimulation with ashift toward T cell reactivity. The mean size of the LN of H&N SCCcontrols was significantly smaller than the control group and those ofthe IRX-2-treated H&N SCC patients were significantly larger than bothcancer and non-cancer control p's <0.01) (FIG. 27A). The non-T and Bcell “other cell” LN area by subtraction and by PAS staining wasapproximately 25% of the total and corresponded mostly to the degree ofsinus histiocytosis (FIG. 5). It is of note that sinus histiocytosis wasmarked in the H&N SCC control but not in the other groups. In 9 of the10 H&N SCC controls, but in none of the other cases, sinus congestionwith erythrocytes was also observed; however, erythrophagocytosis by thehis-lymphocytes, either B and PC or T lymphocytes, were calculated andcorrelated with the area of the lymph node bearing the correspondinglymphoid populations. (FIG. 27C. The T cell area of H&N SCC controls wasmodestly reduced (p=NS) and the density significantly reduced (p <0.01),compared to non-cancer controls (FIG. 27B). T cell area of IRX-2-treatedpatients was modestly increased compared to non-cancer controls (p=NS)and significantly so over the H&N SCC controls (p <0.01). T cell densityof the IRX-2-treatedLN was significantly greater than both controls (p's<0.01) FIG. 27D.

Example 16

FIGS. 28A-D—Sinus histiocytosis is characterized by the majority ofcells being large, granular, PAS-positive and a minority of CD3+ T cellsof varying size. FIG. 28A depicts a typical example of a HN SCC controlwith Sinus Histiocytosis. FIG. 28B shows CD68+ staining of a lymph nodewith Sinus Treatment with IRX-2 was associated with a reversal of sinushistiocytosis apparent in the HN SCC controls. FIG. 28C shows a typicalexample of a lymph node with sinus histiocytosis and erythrocytecongestion. FIG. 28D is a bar graph showing the reversal of sinushistiocytosis in IRX-2 treated patients.

Example 17

FIGS. 29A-F—The upper panel shows examples of the pre-treatment biopsyof three patients with squamous cell head and neck cancer (H&N SCC). Thebiopsies average 80% tumor and 20% stroma with a light sprinkling oflymphocytes in the stroma. The lower panel shows typical sections of thetumor following treatment with the IRX-2 regimen. Notable is the heavyinfiltration of lymphocytes with displacement of tumor. In this trial atINCAN 22/25 patients (88%) showed the response.

Example 18

FIG. 30 Shows the intensity of necrosis and fibrosis in the surgicalspecimen of 11 responder patients (44%) vs 14 non-responder patientstreated with the IRX-2 regimen in the US Phase 2 trial.

Example 19

IRX-2 is shown to stimulate killer T cell infiltration that causes tumordestruction, as shown in FIGS. 31A-D. FIGS. 31A-D are H&E and IHC(Immunohistochemistry) stains of resection specimens from patientstreated with IRX-2. Despite the heterogeneity of tumors, there is seenan increased frequency of the brisk infiltrate pictured in FIG. 31B andnoted by the arrow as a lake of lymphocytes. Pictured in FIG. 31D, theimmunohistochemistry staining confirms that the infiltrate is the CD45ROmemory killer T-cell phenotype. There is growing evidence thatlymphocyte infiltration into tumors predicts improved outcome incolorectal, ovarian, breast, head and neck cancer. The lymphocyticinfiltrate provides a link as to why patients appear to be living longerwithout disease than expected—the result of immune memory that canattack micro metastases and thereby delay or prevent recurrence andimprove survival.

Example 20

Immunohistochemistry was performed to compare intratumoral versusperitumoral infiltrates. Summary data from the evaluation of overallpresence and location of immune cells in the tumor resection specimenbased on immunohistochemistry stain is presented below in Table 12. Thecombined presence of B cells in the tumor's peripheral area and thediffuse intratumoral lymphocytic infiltrate that is primarily CD8+ andCD45 RO+ (ie, the “killer” effector T-cell phenotype) copresent withactivated CD68+ macrophages suggest antigenic stimulation and anantitumor immune response.

TABLE 12 Responders Non-Responders P value CD3 85:15 69:31 <0.05 CD459:41 49:51 NS CD8 93:7  80:20 <0.05 CD45RO 97:3  79:21 <0.01 CD45RA32:68  2:98 <0.02 CD20 30:70 17:83 NS CD68 96:4  88:12 <0.01

Example 21 Dendritic Cells

One of the key cell types that IRX-2 acts on is the dendritic cell.Cancer patients have reduced dendritic cell function as a result ofreduced antigen uptake, antigen presentation, and expression of thesignaling molecules necessary for effective T cell stimulation.

As shown in FIG. 32, the key signaling molecules for T cell stimulationand adhesion on dendritic cells are CD86, CD40 and CD54. In cancerpatients, the components of the antigen presenting machinery aredown-regulated in dendritic cells, resulting in a reduction of effectiveantigen presentation to T cells. IRX-2 is able to activate and maturedendritic cells both phenotypically and functionally. By increasingexpression of the antigen presenting machinery, IRX-2 acts to restoreantigen presentation function.

Shown in FIG. 33 is highly statistically significant flow cytometry datathat show the actions of IRX-2 on the dendritic cell's antigenpresenting machinery and T cell stimulatory capacity. HLA-DRup-regulation is required by dendritic cells to present antigen in theMHC Class II groove. CD86 is the co-stimulatory receptor for naïveT-cells, that is one of the required signals for T-cell activation andthe creation of killer memory T-cells. Dendritic cells that do notexpress CD86 are tolerizing dendritic cells and function to createantigen-specific suppressive regulatory T cells. By administering IRX-2and increasing the co-stimulatory proteins, it is possible to shift thisbalance from tolerizing dendritic cells to activating dendritic cells,and a coordinated and robust immune response against the immune targetis initiated.

FIG. 34 shows highly statistically significant flow cytometry data thatshow the actions of IRX-2 on the dendritic cells' potential T cellstimulatory capacity. Increases in CD40 expression are necessary togenerate a sustained T cell activation and generation of memory T cells.CD54 also called ICAM-1, and is involved in dendritic cell-T cellinteractions and provides a second of the required signals for T-cellactivation. Thus the up-regulation of these molecules in dendritic cellsensures that the cellular immune response initiated by IRX-2 issustained and robust.

FIG. 45 shows data comparing various dilutions of IRX-2 and recombinantTNF and their respective ability to up-regulate CD83 expression, amarker of mature dendritic cells. The multiple active components inIRX-2, present in physiologic quantities, act synergistically toincrease CD83 expression by about 4 times the magnitude of theequivalent amount of TNF present in IRX-2. To achieve similar resultswith TNF alone, 10-25 times the concentration was required, amounts thatwould clearly exceed the concentration in tissue or lymph node(supra-physiologic/pharmacologic).

FIG. 36 shows data from an assay to determine the ability of dendriticcell to induce T-cell proliferation, a functional assessment of adendritic cell's ability to activate naïve T-cells. IRX-2 enhances Tcell stimulatory activity of DC. Immature DC (GM-CSF/IL4×7d) werestimulated with IRX-2 or X-VIVO 10 media alone control (closed or opencircles, respectively). After 48 h, DC were harvested, washedextensively and co-cultured with allogeneic nylon wool-purified T cells(2×105/well) in round bottom 96-well microtiter plates at the indicatedstimulator (DC) to responder (T) ratios. On day 5 of the co-culture,cells were pulsed with BrdU and incorporated BrdU was measured 18 hlater by a colorimetric anti-BrdU ELISA assay. This graph shows theresults from a representative donor of 6 individual donors tested,expressed as mean stimulation index (S.I.) (+/−SEM) at the 4 DC:T ratiostested. S.I. is defined as [(O.D. DC stimulated T cell−O.D. DCalone)/O.D. resting T cell]. The mean S.I. from all 6 donors across theentire range of DC:T ratios showed a statistically significantimprovement when IRX-2-treated DC were used as stimulators (p <.0.05, byANOVA). There was a significant increase in the IRX-2 treated dendriticcells ability to induce T-cell proliferation—a confirmation that thephenotypic dendritic cell changes induced by IRX-2 also results in afunctionally active dendritic cell that is capable of effectivelycausing T cell stimulation and proliferation.

Example 22 IRX-2 Enhances Peptide-Specific IFN-γ Production and DTH

Mice were immunized with varying doses of PSMA peptides with and withoutIRX-2. The T cell response after peptide or conjugate challenge wasassessed by DTH response (FIG. 37A) or IFN-γ production (FIG. 37B) inresponse to a subsequent peptide. IRX-2 plus conjugate vaccine enhancesantigen specific cellular T cell response in vivo (DTH) and ex vivo(IFN-γ production by spleen lymphocytes). This is important because acellular response is an essential requirement in an effective cancervaccine. Also, the T cell responses in vivo and ex vivo are both relatedto the dose of the vaccine used with IRX-2. The dose response confirmsthat the response is vaccine antigen driven and not just due to IRX-2.

Example 23 IRX-2 is Superior to Other Adjuvants in EnhancingPeptide-Specific DTH

The novel nature of the IRX-2 activity was confirmed by comparing IRX-2to other adjuvants which were selected to represent various mechanismsof action. Alum was evaluated because it is a widely used FDA approvedadjuvant, CpG because it is a TLR agonist that targets antigenpresenting cells and the RIBI Adjuvant System (RAS) because it containsmultiple adjuvant activities and is a safer alternative than Freund'sadjuvant. As shown in FIG. 38 all of the adjuvants tested caused a DTHresponse when challenged with the conjugate; however, only IRX-2enhanced the peptide-specific DTH response to the conjugate vaccinewhile alum, CpG or RAS did not as shown in FIG. 43. The studies reportedhere provide important preclinical data supporting the hypothesis thatIRX-2 enhances T cell immune responses to exogenous antigens for use incombination with multiple antigen types in therapeutic cancer vaccines.The unique nature of the T cell peptide-specific response to theconjugate vaccine is a result of the multi-target mode of action ofIRX-2 and the presumed synergy among the cytokines.

Example 24 Evidence of Action on Immune Cells in Peripheral Circulation

The following data are summarized from immune monitoring of peripheralblood at baseline and day 21. Statistically significant decreases inperipheral blood in 21 days in the CCR7⁺ cell populations-those withlymph node homing potential and B cells are consistent with these cellsrecruitment out of the circulation and into lymph nodes for activationby dendritic cells. No change or a slight trend towards increasednumbers of effector cells are consistent with cytotoxic T cells thatpass temporarily into the circulation and then into tissue to kill theantigenic target. The overall changes in peripheral blood of the IRX-2regimen treated patients of both immune cells and T regulatory cells areconsistent with an immunization in lymph nodes a shift from a tolerizingto a stimulating environment, as shown generally in FIG. 39.

Presented in FIG. 40 are the absolute counts at baseline and day 21(after completion of IRX-2 regimen) of T regulatory cells in peripheralblood from Head and Neck cancer patients. Each line represents a patientand the bold line represents the group mean. Several prior studies haveindicated that T reg cells are increased in cancer patients (ovarian,colorectal, hepatocellular, HNSCC) and that increased T regs isassociated with a worse prognosis. The fact that the T regs of 18 of 26patients stay the same or go down in only 21 days is a striking andsignificant finding because tolerizing dendritic cells should continueto expand the T reg population. The IRX-2 regimen stabilizes Treg countsat baseline levels is a significant finding reflective of improvedsurvival in these patients.

Example 25 Evidence of Tumor Shrinkage Consistent with Immunization

FIGS. 41A and 41B show fused FDG-PET/CT scans of a large 5.2 cm rightbase of tongue primary with two involved lymph nodes shows a 58%reduction in volume and a 75% reduction in total glycolytic activity in21 days. There is evidence of tumor shrinkage which supports thehypothesis that an anti-tumor rejection and immunization is occurring.

Example 26

Previously, the criteria for histopathology of a biopsy versus a tumorspecimen (Meneses) were that the tumor was reduced overall,fragmentation of the tumor occurred, and there was increased lymphocyteinfiltration (LI). According to the present invention, there are newcriteria presented herein for a treated tumor versus a control tumor,namely tumor disruption with necrosis and fibrosis, and increased LIthat is greater intratumorally than peritumorally. Table 13 belowsummarizes various findings of cytokine treatment on H&NSCC.Importantly, IRX-2 is shown to work on all arms of the immune systemwhereas other multiple component cytokine therapeutics do not. MULTIKINE(Cel-Sci) includes multiple cytokines in its formulation; however, itseffect is a single one on the tumor itself, not on the immune system.

TABLE 13 Treated Control De Stefani Tumor Control ↑ LI, ↑ necrosis, ↑fibrosis rIL-2 tumor Meneses Tumor Biopsy ↑ LI, ↓ tumor, ↑fragmentationIRX-2 Feinmesser Tumor Biopsy ↑ LI, ↓ tumor Multikine Timar TumorControl ↑ LI, No ↓ tumor or fragmentation Multikine tumor IRX TumorBiopsy ↑ LI - small tumor, ↑fragmentation Therapeutics Tumor Control ↑LI, ↑ fibrosis tumor

OVERALL CONCLUSION

This study confirms and extends Applicants' prior observationsconcerning the ability of the IRX-2 regimen to have significantbiological activity on patients with squamous cell head and neck cancertreatment prior to surgery. The present study confirms that thetreatment is safe with few adverse events attributed to the regimen. Infact, those patients who showed evidence of histopathologic changes oflymphocyte infiltration had the majority of symptom improvements likereduced pain and tenderness, improved breathing and phonation, andsoftening of the tumor (as sign of dissolution). Three patients wereadjudged to have clinical responses (2PRs, 1MR). Overall survival dataand recurrence free survival while immature are encouraging and similarin degree and profile to Applicants' previous study. Notable is that nodeaths occurred due to recurrence in the first 12 months of follow up.All deaths to date but one are in the non-responder group.

The most compelling data are those associated with the mechanism ofaction studies. It was observed that declines of B lymphocytes and two Tcell subsets associated with initial immunization and lymph node homing.No increases in memory/effector cell were observed in blood; however,this is explainable based upon the traffic patterns of T cells whichoccur with an immunization. Notably no increase in T regs was observed.

Applicants' prior studies showed that patients responding to the IRX-2regimen show increase of uninvolved lymph nodes proximal to the tumor,replenishment of depleted T lymphocyte areas and the picture ofactivation as occurs with antigen. Thus, lymphocytes are trafficking viablood and lymphatics to the regional lymph nodes where they arepresumably immunized to autologous tumor antigens. As shown herein, theythen leave the lymph node and travel by blood to the tumor where theyinfiltrate in and around the tumor and correlate with evidence of tumordestruction (necrosis, fibrosis, and tumor reduction). In the patientsshowing this reaction, the increases in lymphocyte infiltration involvespredominantly CD3+ CD4+ CD45RA+ T cell populations and CD20+ Blymphocytes around the tumor periphery and CD3+ CD8+ CD45RO+ Tlymphocyte populations and macrophages within the tumor. The changeswithin the tumor are greater than these in the periphery. This mechanismis generally shown in FIG. 2.

Notably, untreated patients show such a reaction only occasionally (20%)and while significantly less frequently than patients treated with theIRX-2 regimen (44% vs. 20%) the presence of the reaction in controlsrepresent a new biomarker for predicting favorable outcome.

The picture is an integrated one clinically, radiologically,pathologically, and immunologically and provides ample evidence for animmunization to autologous tumor antigen. IRX-2 is shown to activate allarms of the immune system to provide a total restoration of immunefunction and ability to attack immune targets.

Throughout this application, various publications, including UnitedStates patents, are referenced by author and year and patents by number.Full citations for the publications are listed below. The disclosures ofthese publications and patents in their entireties are herebyincorporated by reference into this application in order to more fullydescribe the state of the art to which this invention pertains.

The invention has been described in an illustrative manner, and it is tobe understood that the terminology which has been used is intended to bein the nature of words of description rather than of limitation.

Obviously, many modifications and variations of the present inventionare possible in light of the above teachings. It is, therefore, to beunderstood that within the scope of the appended claims, the inventionmay be practiced otherwise than as specifically described.

REFERENCES

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1. A method of treating an immune target in a patient, including thesteps of: administering an effective amount of a primary cell derivedbiologic to the patient; inducing immune production; blocking immunedestruction; and treating the immune target in the patient.
 2. A methodof treating a tumor in a patient, including the steps of: administeringan effective amount of a primary cell derived biologic to the patient;inducing immune production; blocking immune destruction; and treatingthe tumor in the patient.
 3. A method of immune prophylaxis, includingthe steps of: administering an effective amount of a primary cellderived biologic to a patient; inducing immune production; blockingimmune destruction; and preventing immune suppression in the patient. 4.A method of preventing tumor escape in a patient, including the stepsof: administering an effective amount of a primary cell derived biologicto the patient; inducing immune production; blocking immune destruction;and preventing tumor escape in the patient.